Method of detecting microorganisms

ABSTRACT

The present invention relates generally to a method for detecting, enumerating and/or identifying microorganisms in a sample. More particularly, the present invention provides a method for determining total microbial content in a sample by detecting the presence of nucleotide sequences associated with all or part of 16S rDNA or its corresponding 16S rRNA or its homologue, functional equivalent or derivative. The nucleotide sequences of the present invention may be used as an indicator of any microorganism and, hence, represents a universal target sequence which is indicative of total microbial content in a sample. The universal target sequence may also be varied to render same genus or species specific or the universal target used to trap microbial DNA or RNA which may be subsequently analyzed by sequence analysis or genetic probe technology. The universal target sequence is useful inter alia to design as universal primers and probes to amplify any microbial-derived genomic sequence, as a means to detect and enumerate total microorganisms and to identify microorganisms in a sample at the genus or species level. Such uses enable improved methods of enviroprotection, bioremediation, medical diagnosis and industrial microbiology. The present invention further relates to the universal target sequence in isolated form and/or primers or probes capable of hybridizing to same and kits for the detection of total microbial content in a sample.

This is a U.S. National Phase under 35 U.S.C. §371 of InternationalApplication PCT/AU01/00933, filed Jul. 27, 2001, which claims priorityto Australian Provisional Patent Application No. PQ9090, filed Jul. 28,2000.

FIELD OF THE INVENTION

The present invention relates generally to a method for detecting,enumerating and/or identifying microorganisms in a sample. Moreparticularly, the present invention provides a method for determiningtotal microbial content in a sample by detecting the presence ofnucleotide sequences associated with all or part of 16S rDNA or itscorresponding 16S rRNA or its homologue, functional equivalent orderivative. The nucleotide sequences of the present invention may beused as an indicator of any microorganism and, hence, represents auniversal target sequence which is indicative of total microbial contentin a sample. The universal target sequence may also be varied to rendersame genus or species specific or the universal target used to trapmicrobial DNA or RNA which may be subsequently analyzed by sequenceanalysis or genetic probe technology. The universal target sequence isuseful inter alia to design as universal primers and probes to amplifyany microbial-derived genomic sequence, as a means to detect andenumerate total microorganisms and to identify microorganisms in asample at the genus or species level. Such uses enable improved methodsof enviroprotection, bioremediation, medical diagnosis and industrialmicrobiology. The present invention further relates to the universaltarget sequence in isolated form and/or primers or probes capable ofhybridizing to same and kits for the detection of total microbialcontent in a sample.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in thisspecification are collected at the end of the description.

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in Australia or any othercountry.

The rapidly increasing sophistication of DNA technology is greatlyfacilitating research and development in a range of disciplinesincluding the medical and allied health industries, the agricultural andhorticultural sectors and in the screening of related genomic sequencesin environmental samples. Of particular importance is the application ofmolecular approaches to the characterization of bacterial communities.Such approaches overcome the limitations imposed by culture-mediatedtechniques for detecting microorganisms. It is known that theunculturable fraction of a microbial population represents a majorcomponent of all microbial communities (1, 2, 3).

Culture dependent methods for enumerating bacterial numbers are known tobe biased since bacteria can only be cultivated if their metabolic andphysiological requirements can be reproduced in vitro. These techniquesmay take several days to yield a result and, therefore, areinappropriate in situations where rapid diagnostic decisions arerequired. Where complex fastidious microbial communities are underinvestigation, such as the variety of microbial habitats in the oralcavity, enumerating bacteria by traditional microbial culturingtechniques may also produce erroneous results.

Fluorescence-based methods for detecting bacteria can also be used toenumerate bacteria. For instance, flow cytometry can be applied to therapid and automated counting of pure cultures used in industrialapplications such as the food and biotechnology industries. However,most bacteria are optically too similar to resolve from each other orfrom debris using flow cytometry, without artificially modifying thetarget bacteria using fluorescent labelling techniques such asfluorescent antibodies or fluorescent dyes (4). The fluorescent DNAstain, diamidinopheylindole (5), for example, can be used to enumeratecomplex bacterial populations. However, differences in bacterial cellsize, coaggregation of bacteria and the presence of differentcontaminating matrices (e.g. mud, food, dental plaque, dentine) can makemeaningful counting difficult if not problematic as it can with director fluorescence microscopy (4).

Rapid enumeration of bacteria can also be achieved using a variety ofmolecular approaches (1, 2, 3, 6). Generally, however, multiple primersare required to detect the bacteria of interest. Techniques, such ascompetitive PCR (7, 8), are labour intensive and require the analysis ofresults from multiple reactions for each test sample. There is a need,therefore, to develop improved molecular approaches to microbialdetection and enumeration.

Real-time PCR such as the TaqMan (Registered trade mark) systemdeveloped by Applied Biosystems relies on the release and detection of afluorogenic probe during each round of DNA amplification. It allows forthe rapid detection and quantification of DNA without the need forpost-PCR processing such as gel electrophoresis and radioactivehybridization (9). In addition, the built-in 96 well format greatlyincreases the number of samples that can be simultaneously analyzed. Themethod uses the 5′ exonuclease activity of a Taq polymerase (AmpliTaqGold, PE Biosystems (Foster City, Calif., USA) during primer extensionto cleave a dual-labelled, fluorogenic probe hybridized to the targetDNA between the PCR primers. Prior to cleavage, a reporter dye, such as6-carboxyfluorescein (6-FAM) at the 5′ end of the probe is quenched by6-carboxy-tetramethylrhodamine (TAMRA) through fluorescent resonanceenergy transfer. Following digestion, FAM is released. The resultingfluorescence is continuously measured in real-time at 518 nm during thelog phase of product accumulation and is proportional to the number ofcopies of the target sequence.

In work leading up to the present invention, the inventors developed aset of oligonucleotides in the form of primers and probes whichuniversally permit detection and quantification of the total bacterialload within a sample. The primers and probes are directed to 16S rDNA orits 16S rRNA and are conveniently used with real-time PCR or similar orrelated technology to detect and enumerate any microorganism not being aEucarya or Archea. The development of a universal primer-probe setpermits the rapid and accurate determination of microbial load withoutnecessitating the development of specific primers for particularmicroorganisms. However, such specific primers may additionally be usedto identify microorganisms at the genus or species level. The presentinvention further provides nucleic and extraction procedures usefulinter alia in screening total biota for the presence of microorganisms.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1, <400>2, etc. A sequence listing isprovided after the claims.

The present invention provides the design and evaluation of a set ofuniversal primers and probes for the amplification of 16S rDNA or 16SrRNA from microorganisms to estimate total bacterial load by inter aliaReal-Time PCR or similar or related technology. The universal primersand probes enable broad specificity in terms of the range ofmicroorganisms which can be detected whilst not detecting Eucarya orArchea. A DNA standard representing those bacteria most likely topredominate in a given habitat is useful to more accurately determinetotal bacterial load. The universal primers and probes for totalmicrobial-derived genomic material can be modified to enableidentification and enumeration of microbial genera or species.Alternatively, or in addition, the universal primers/probes may be usedas a trap for microbial 16S rDNA or 16S rRNA which may then be sequencedor interrogated by genus or species specific probes or primers. Anucleic acid extraction procedure is also provided in accordance withthe present invention. The universal primers and probes have a range ofuses in the medical, agricultural and other commercial industries.

Accordingly, one aspect of the present invention contemplates a methodfor determining total microbial content in a sample, said methodcomprising amplifying a target nucleotide sequence which issubstantially conserved amongst two or more species of microorganisms,said amplification being for a time and under conditions sufficient togenerate a level of an amplification product which is proportional tothe level of microorganisms in said sample.

Another aspect of the present invention provides a method fordetermining total microbial content in a sample, said method comprisingamplifying a target nucleotide sequence comprising or associated with16S rDNA or 16S rRNA or a homologue or derivative or functionalequivalent thereof, said amplification being for a time and underconditions sufficient to generate a level of an amplification productwhich is proportional to the level of microorganisms in said sample.

Yet another aspect of the present invention is directed to a method fordetermining total microbial content in a sample, said method comprisingsubjecting a nucleotide sequence defining or associated with 16S rDNA or16S rRNA to Real-Time PCR or equivalent technology for a time and underconditions to generate a level of amplification product which isproportional to the level of microorganisms in said sample.

Still another aspect of the present invention provides a complexcomprising forward and reverse primers hybridized to complementarystrands of a target sequence comprising all or part of 16S rDNA or 16SrRNA or a homologue or derivative or functional equivalent thereof andan oligonucleotide probe labelled at its 5′ end by a fluorogenicreporter molecule and at its 3′ end by a molecule capable of quenchingsaid fluorogenic molecule, said oligonucleotide probe hybridized to aportion of said 16S rDNA or 16S rRNA which is nested between saidforward and reverse primers.

Even yet another aspect of the present invention contemplates a methodfor determining the total microbial content in a sample, said methodcomprising subjecting DNA in said sample to Real-Time PCR using aprimers-probe set which comprises primers selected to amplify DNAcomprising or associated with 16S rDNA or 16S rRNA or a homologue orderivative or functional equivalent thereof and a probe which hybridizesto a nucleotide sequence nested between said primers wherein said probeis labelled at its 5′ end by a fluorogenic reporter molecule and at its3′ end by a molecule capable of quenching said fluorogenic molecule,said amplification being for a time and under conditions to generate alevel of amplification product which is proportional to the level ofmicroorganisms in said sample.

Still another aspect of the present invention provides a method foridentifying a particular microorganism or prevalence of a particulargenus or species of microorganism in a sample, said method comprisingcapturing DNA or RNA in said sample by primer(s) having a nucleotidesequence complementary to a nucleotide sequence within 16S rDNA or 16SrRNA and then subjecting said captured DNA or RNA to nucleotidesequencing and/or interrogation by a genus or species specific probe andthen determining the microorganism by the particular sequence or patternof probe interrogation.

Even still another aspect of the present invention is directed to a kitin compartmental form, said kit comprising a compartment adapted tocontain one or more primers capable of participating in an amplificationreaction of DNA comprising or associated with 16S rDNA or 16S rRNA,another compartment comprising a probe labelled at its 5′ end by afluorogenic reporter molecule and at its 3′ end by a molecule capable ofquenching said fluorogenic molecule and optionally another compartmentadapted to contain reagents to conduct an amplification reaction andoptionally a compartment adapted for extraction of nucleic acid fromcells.

A further aspect of the present invention contemplates a method forextracting nucleic acid material from a sample comprising microbialcells, said method comprising subjecting a concentrated sample of saidcells to enzymatic degradation and lysing said cells in the presence ofSDS and then purifying said nucleic acid material.

Another aspect of the present invention further provides a method forextracting nucleic acid material from a sample comprising microbialcells, said method comprising subjecting a concentrated sample of saidcells to pressure-mediated disruption, enzymatic degradation and thenlysing said cells in the presence of SDS and then purifying said nucleicacid material.

Yet another aspect of the present invention contemplates a method fordetermining microorganisms in a sample, said method comprising:

-   -   optionally subjecting a concentrated sample of said cells to        pressure-mediated disruption followed by enzymatic degradation        and then lysing said cells in the presence of SDS and then        purifying said nucleic acid material;    -   amplifying said nucleic acid material in the presence of forward        and reverse primers capable of hybridizing to a conserved        nucleotide sequence within 16S rDNA or 16S rRNA;    -   optionally detecting the presence of amplified product in the        presence of a probe labelled with a reporter molecule and        determining the total microbial content; and        optionally isolating the amplified product and either sequencing        the isolated product or subjecting the amplified product to        genetic interrogation to identify the genus or species of        microorganism present.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a representation showing conservation of sequences used in theuniversal primer-probe set from the 16S rDNAs of bacteria representingmost of the groups of Procarya defined in Bergey's Manual ofDeterminative Bacteriology (12). (A) Alignment of rDNAs showingconservation of 19 bp forward primer (outlined in bold). The bacterialsequences are designated SEQ ID NOS: 10-41. (B) Alignment of rDNAsshowing conservation of 23 bp probe sequence (outlined in bold) Thebacterial sequences are designated SEQ ID NOS: 42-71. (C) Alignment ofrDNAs showing conservation of 26 bp reverse primer (outlined in bold).The bacterial sequences are designated SEQ ID NOS: 72-106.

FIG. 2 is a graphical representation showing the standard curve using E.coli DNA.

FIG. 3 is a graphical representation showing the sensitivity of theuniversal probe and primers in detecting E. coli DNA using Real-TimePCR. Purified E. coli DNA was used as the template in quantities 2380pg, 238 pg, 23.8 pg, 2.38 pg, 238 fg, 23.8 fg, representing C_(T)(threshold cycle) values in the range 16.9 to 36.3 where the interceptof the magnitude of the fluorescent signal (ΔR_(n)) with the horizontalthreshold line in bold represents the C_(T) value for a given sample.The fluorescent signal at C_(T) 37.7 corresponds to theno-template-control and represents bacterial DNA contamination in thecommercially supplied reagents.

FIG. 4 is a graphical representation showing the effect of sonication ofbacterial cells on the isolation of DNA.

FIG. 5A is a photographic representation showing the presence ofnucleases in P. gingivalis. (1) Freeze/thawed sample; (2)Freeze/thawed-boiled sample; (3) Freeze/thawed sample treated withmutanolysin; (4) Freeze/thawed-boiled sample treated with mutanolysin;(5) Sample sonicated for 3 min; (6) Sample sonicated for 6 min; (7)Sample sonicated for 3 min and treated with mutanolysin; and (8) Samplesonicated for 6 min and treated with mutanolysin.

FIG. 5B is a photographic representation showing degradation of DNA byfreeze/thawed sample of P. gingivalis. (1) Fusobacterium nucleatum DNA;(2) Lactobacillus acidophilus DNA; (3) Porphyromonas gingivalis DNA; (4)Prevotella melaninogenica DNA; (5) Streptococcus mutans DNA; (6)Peptostreptococcus micros DNA; (7) Porphyromonas endodontalis DNA; and(8) Escherichia coli DNA.

FIG. 6A is a graphical representation showing the critical role ofnucleases and the effect of ZnCl₂ on the quantification of P. gingivalisand P. gingivalis+S. mutans.

FIG. 6B is a graphical representation showing the critical role ofnucleases and the effect of ZnCl₂ on the quantification of P. gingivalisand P. gingivalis+E. coli.

FIG. 7 is a graphical representation showing the effect of removal ofZnCl₂ and sodium dodecyl sulphate (SDS) on the quantification of DNAusing undiluted samples.

FIG. 8 is a graphical representation showing the internal positivecontrol using B. tryoni dsX gene insert in pGEM (registered trademark)-T Easy vector system.

FIG. 9A is a photographic representation showing isolation of DNA usingATL buffer and two-step DEPC method: bacteria identified asStreptococci. (1) S. mitis using ATL buffer; (2) S. intermedius usingATL buffer; (3) S. intermedius using ATL buffer; (4) S. costellatususing ATL buffer; (5) S. mitis using two-step DEPC method; (6) S.intermedius using two-step DEPC method; (7) S. intermedius usingtwo-step DEPC method; and (8) S. costellatus using two-step DEPC method.

FIG. 9B is a photographic representation showing isolation of DNA usingATL buffer and two-step DEPC method: bacteria identified as Actinomyces.(1) A. viscosus by ATL method; (2) A. viscosus by two-step DEPC method;(3) A. georgiae by ATL method; and (4) A. georgiae by two-step DEPCmethod.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated in part on the identification of anucleotide sequence associated with or comprising the 16S rDNA or 16SrRNA or its homologue, functional equivalent or derivative which isconserved amongst all prokaryotic microorganisms. The identification ofthese conserved nucleotide sequences enables the detection andquantification of total microbial content in a sample. The term“functional equivalent” in this context includes other conservedsequences which may also be used to determine total microbial content.The present invention provides primers and probes based on theseconserved sequences which are “universal” in the sense that they arecapable of hybridizing and/or amplifying microbial nucleic acidmolecules without substantial cross reaction with DNA from Eucarya orArchea. The universal primers or probes may also be modified to rendersame genus or species specific or used in conjunction with other genus-or species-specific primers or probes such as to interrogate amplifiednucleic acid material. The universal primers and probes may also be usedas a “trap” for prokaryotic nucleic acid material which may inter aliabe sequenced to assist in identifying a particular microorganism or todetermine the prevalence of a particular microorganism at the genus orspecies level.

Accordingly, one aspect of the present invention contemplates a methodfor determining total microbial content in a sample, said methodcomprising amplifying a target nucleotide sequence which issubstantially conserved amongst two or more species of microorganisms,said amplification being for a time and under conditions sufficient togenerate a level of an amplification product which is proportional tothe level of microorganisms in said sample.

Reference to “determining” microbial content includes estimating,quantifying, calculating or otherwise deriving a level of microbialcontent. The level of microbial content is generally referred to as thetotal microbial content and includes microorganisms which are capable ofbeing cultured as well as microorganisms which cannot be cultured. Thelevel of total microbial content is conveniently expressed in terms ofnumber of microbial cells per particular volume, wet or dry weight ofmicrobial cells per particular volume or other suitable indicator of thetotal number of cells in a sample. Conveniently, the number of cells isexpressed per millilitre, per microlitre or per 25 or 50 microlitres.The number of microorganisms may also be determined indirectly such ascorresponding to a particular amount of DNA. For example, 0.496 picogramof E. coli DNA corresponds to approximately 100 E. coli cells in thesample. The term “determining” may also be identifying a particularmicroorganism or ascertaining the prevalence of a particularmicroorganism at the genus or species level. This may, for example, beaccomplished by nucleotide sequence and/or nucleic acid interrogation byspecies- or genus-specific probes.

The term “microorganism” is used in its broadest sense and includes Gramnegative aerobic bacteria, Gram positive aerobic bacteria, Gram negativemicroaerophillic bacteria, Gram positive microaerophillic bacteria, Gramnegative facultative anaerobic bacteria, Gram positive facultativeanaerobic bacteria, Gram negative anaerobic bacteria, Gram positiveanaerobic bacteria, Gram positive asporogenic bacteria andActinomycetes. Conveniently, reference herein to a microorganismincludes a member of the group of Procarya as listed in Bergey's Manualof Determinative Bacteriology (12). The term “microorganism” or“microbial” generally pertains to a bacterium or bacterial and which isnot a member of Eucarya or Archea.

Although the present invention is particularly directed to thosemicroorganisms listed in Table 3, the present invention extends to anymicrobial cell which carries the conserved target nucleotide sequence.

The term “sample” is used in its broadest sense to include biological,medical, agricultural, industrial and environmental samples. Forexample, samples may be derived from culture fluid, biopsy fluid ortissue from human, animal or insect sources, samples from naturalenvironments such as soil, river, hot mineral water springs, plant,antarctic, air or extraterrestrial samples as well as samples fromindustrial sites such as waste sites and areas of oil spills or aromaticor complex molecule contamination and pesticide contamination. Thesample may also comprise food, food components, food derivatives and/orfood ingredients including food products formed in the dairy industrysuch as milk. The sample may be liquid, solid, slurry, air, vapour,droplet or aerosol or a combination of any of the above.

The target nucleotide sequence is generally a target DNA or RNAsequence. If the target is an RNA sequence, then this sequence may haveto be subject to reverse transcription to generate a complementary DNAsequence (cDNA). Conveniently, the target nucleotide sequence is DNA andis conserved amongst two or more species of microorganisms. In aparticularly preferred embodiment, the target sequence is ribosomal DNA(rDNA) such as but not limited to 16S rDNA or is ribosomal RNA (rRNA)such as but not limited to 16S rRNA. With respect to the latter,suitable microbial cells are any cells which comprise a conservedsequence comprising or associated with 16S rDNA or 16S rRNA. Referenceherein to “16S rDNA” or “16S rRNA” includes reference to any homologuesor derivatives thereof as well as functional equivalents thereof A“homologue” of 16S rDNA includes RNA forms such as 16S rRNA or viceversa.

Accordingly, a preferred aspect of the present invention provides amethod for determining total microbial content in a sample, said methodcomprising amplifying a target nucleotide sequence comprising orassociated with 16S rDNA or 16S rRNA or a homologue or derivative orfunctional equivalent thereof, said amplification being for a time andunder conditions sufficient to generate a level of an amplificationproduct which is proportional to the level of microorganisms in saidsample.

Although the present invention may be practised directly on singlestranded template from a non-amplified nucleic acid molecule, in apreferred embodiment the template nucleic acid molecule is from anucleic acid molecule which has been subjected to amplification. Any ofa range of amplification reactions may be employed including PCR,rolling circle amplification and Qβ replicase based amplificationamongst others.

The preferred amplification conditions are those which result inreal-time Real-Time PCR. The amplification product is then measured to aparticular amount referred to herein as the threshold concentration(C_(T)). The C_(T) is proportional to the total target sequence (e.g.16S rDNA) and hence proportional to total bacterial content. Generally astandard curve is prepared based on the C_(T) and known amounts of DNAin pg by determining the level of amplification product under conditionsgiving a C_(T), this then determines the amount of microbial targetsequence and, hence, microbial levels. The use of Real-Time PCR ispreferred but the present invention permits the use of relatedtechnology.

Accordingly, another aspect of the present invention is directed to amethod for determining total microbial content in a sample, said methodcomprising subjecting a nucleotide sequence defining or associated with16S rDNA or 16S rRNA or a homologue or derivative or functionalequivalent thereof to Real-Time PCR for a time and under conditions togenerate a level of amplification product which is proportional to thelevel of microorganisms in said sample.

Preferably, the level of amplification product is defined by C_(T).

The time and conditions for amplification such as Real-Time PCR is suchthat, in a preferred embodiment, C_(T) is recorded. These conditions arethe same as for preparation of a standard curve.

In a particularly preferred embodiment, the amplification is conductedwith a set of primers (forward and reverse) and a probe oligonucleotidelabelled with a fluorogenic reporter molecule at its 5′ end and aquenching molecule at its 3′ end. The quenching molecule preventsemission of signal from the fluorogenic reporter molecule. The probeoligonucleotide hybridizes to a region of the target sequence betweenthe regions to which the forward and reverse primers hybridize. As thepolymerase moves along the strand to which the probe oligonucleotide hashybridized, the 5′ end of the probe is cleaved off by the exonucleaseactivity of the polymerase thus permitting emission of the fluorogenicsignal due to separation of the quenching moiety.

In another embodiment, therefore, the present invention provides acomplex comprising forward and reverse primers hybridized tocomplementary strands of a target sequence comprising all or part of 16SrDNA or 16S rRNA or a homologue or derivative or functional equivalentthereof and an oligonucleotide probe labelled at its 5′ end by afluorogenic reporter molecule and at its 3′ end by a molecule capable ofquenching said fluorogenic molecule, said oligonucleotide probehybridized to a portion of said 16S rDNA which is nested between saidforward and reverse primers.

The preferred primers and probes of the present invention exhibit atleast one of the following properties:

-   (i) comprise a melting temperature (T_(m)) of DNA between about    58° C. and about 60° C. for primers and about 68° C. and 70° C. for    the probe;-   (ii) comprise a GC content of between about 30 and 80%;-   (iii) comprise no more than three consecutive G's in the primer or    probe;-   (iv) comprise no more than 2 GC's in the last 5 nucleotides at the    3′ end of the primer;-   (v) comprise no G on the 5′ end of the probe;-   (vi) the selection of probe should be from the strand with more C's    than G's; and-   (vii) the amplicon length should be between about 50 and about 150    bp.

In a most preferred embodiment, primers-probe set are as follows:

Universal forward primer: TCCTACGGGAGGCAGGAGT (SEQ ID NO:1) Universalreverse primer: GGACTACCAGGGTATCTAATCCTGTT (SEQ ID NO:2) Universalprobe: CGTATTACCGCGGCTGCTGGCAC. (SEQ ID NO:3)

Accordingly, another aspect of the present invention contemplates amethod for determining the total microbial content in a sample, saidmethod comprising subjecting DNA in said sample to Real-Time PCR using aprimers-probe set which comprise primers selected to amplify DNAcomprising or associated with 16S rDNA or 16S rRNA or a homologue orderivative or functional equivalent thereof and a probe which hybridizesto a nucleotide sequence nested between said primers wherein said probeis labelled at its 5′ end by a fluorogenic reporter molecule and at its3′ end by a molecule capable of quenching said fluorogenic molecule,said amplification being for a time and under conditions to generate alevel of amplification product which is proportional to the level ofmicroorganisms in said sample.

Preferably, the forward and reverse primers and probe are those definedby SEQ ID NO:SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively orforward and reverse primers and probe which hybridize to a complementaryform of SEQ ED NO:1, SEQ ID NO:2 or SEQ ID NO:3, respectively under lowstringency conditions and/or which exhibit at least about 70% similarityto SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 or their complementary forms.The probe is conveniently labelled at its 5′ end with a reportermolecule such as but not limited to a fluorescent dye, for example,6-carboxyfluorescein (6-FAM). The 3′ end is conveniently labelled with aquenching molecule such as but not limited to6-carboxy-tetramethylrhodamine (TAMRA).

The term “similarity” as used herein includes exact identity betweencompared sequences at the nucleotide level. In a particularly preferredembodiment, nucleotide sequence comparisons are made at the level ofidentity rather than similarity.

Terms used to describe sequence relationships between two or morepolynucleotides include “reference sequence”, “comparison window”,“sequence similarity”, “sequence identity”, “percentage of sequencesimilarity”, “percentage of sequence identity”, “substantially similar”and “substantial identity”. A “reference sequence” is at least 12 butfrequently 15 to 18 monomer units in length. Because two polynucleotidesmay each comprise (1) a sequence (i.e. only a portion of the completepolynucleotide sequence) that is similar between the twopolynucleotides, and (2) a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window” refers to aconceptual segment of typically 12 contiguous nucleotides that iscompared to a reference sequence. The comparison window may compriseadditions or deletions (i.e. gaps) of about 20% or less as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. Optimal alignment ofsequences for aligning a comparison window may be conducted bycomputerized implementations of algorithms (GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package Release 7.0, GeneticsComputer Group, 575 Science Drive Madison, Wis., USA) or by inspectionand the best alignment (i.e. resulting in the highest percentagehomology over the comparison window) generated by any of the variousmethods selected. Reference also may be made to the BLAST family ofprograms as for example disclosed by Altschul et al. (18). A detaileddiscussion of sequence analysis can be found in Unit 19.3 of Ausubel etal. (19).

The terms “sequence similarity” and “sequence identity” as used hereinrefers to the extent that sequences are identical or functionally orstructurally similar on a nucleotide-by-nucleotide basis over a windowof comparison. Thus, a “percentage of sequence identity”, for example,is calculated by comparing two optimally aligned sequences over thewindow of comparison, determining the number of positions at which theidentical nucleic acid base (e.g. A, T, C, G, I) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity. For the purposes of thepresent invention, “sequence identity” will be understood to mean the“match percentage” calculated by, for example, GAP in the WisconsinGenetics Software Package or other programs such as the DNASIS computerprogram (Version 2.5 for windows; available from Hitachi Softwareengineering Co., Ltd., South San Francisco, Calif., USA) using standarddefaults as used in the reference manual accompanying the software.Similar comments apply in relation to sequence similarity.

Reference herein to a low stringency includes and encompasses from atleast about 0 to at least about 15% v/v formamide and from at leastabout 1 M to at least about 2 M salt for hybridization, and at leastabout 1 M to at least about 2 M salt for washing conditions. Generally,low stringency is at least from about 25-30° C. to about 42° C. Thetemperature may be altered and higher temperatures used to replaceformamide and/or to give alternative stringency conditions. Alternativestringency conditions may be applied where necessary, such as mediumstringency, which includes and encompasses from at least about 16% v/vto at least about 30% v/v formamide and from at least about 0.5 M to atleast about 0.9 M salt for hybridization, and at least about 0.5 M to atleast about 0.9 M salt for washing conditions, or high stringency, whichincludes and encompasses from at least about 31% v/v to at least about50% v/v formamide and from at least about 0.01 M to at least about 0.15M salt for hybridization, and at least about 0.01 M to at least about0.15 M salt for washing conditions. In general, washing is carried outT_(m)=69.3+0.41 (G+C) % (20). However, the T_(m) of a duplex DNAdecreases by 1° C. with every increase of 1% in the number of mismatchbase pairs (21). Formamide is optional in these hybridizationconditions. Accordingly, particularly preferred levels of stringency aredefined as follows: low stringency is 6×(SSC) buffer, 0.1% w/v sodiumdodecyl sulphate (SDS) at 25-42° C.; a moderate stringency is 2×SSCbuffer, 0.1% w/v SDS at a temperature in the range 20° C. to 65° C.;high stringency is 0.1×SSC buffer, 0.1% w/v SDS at a temperature of atleast 65° C.

The primers and probes may be modified to render same genus- orspecies-specific. Alternatively, or in addition, further primers orprobes may be employed to specifically define a genus or species ofmicroorganism by, for example, primer/probe interrogation. With respectto the former, the universal primer/probe set may be used as a trap for16S rDNA/rRNA or its homologues, equivalents or derivatives which isthen subjected to identification of genus or species of themicroorganism or of the predominant microorganism. Some partialpreselection may also be conducted to bias the sample to, for example,particular types of microorganisms such as aerobes, anaerobes ormicrobes having particular nutritional requirements or features orantibiotic-resistance microbes.

Accordingly, another aspect of the present invention contemplates amethod for identifying a particular microorganism or prevalence of aparticular genus or species of microorganism in a sample, said methodcomprising capturing DNA or RNA in said sample to primer having anucleotide sequence complementary to a nucleotide sequence within 16SrDNA or 16S rRNA and then subjecting said captured DNA or RNA tonucleotide sequencing and/or interrogation by a genus or speciesspecific probe and then determining the microorganism by the particularsequence or pattern of probe interrogation.

In a related embodiment, there is provided a method for identifying amicroorganism by its genus in a sample, said method comprisingsubjecting DNA in said sample to Real-Time PCR using a primers-probe setwhich comprises primers selected to amplify DNA comprising or associatedwith 16S rDNA or 16S rRNA and a probe which hybridizes to a nucleotidesequence nested between said primers wherein said probe is eitherspecific for said microorganism to be identified or which issubsequently identified by a genus-specific probe.

In a preferred embodiment, the primer is also a genus-specific probe.

In one particularly useful embodiment, the primer/probe set is used totrap nucleic acid material which is then cloned and sequenced todetermine the genus or species of the predominant microbe. A decisionmay then be made to study or cultivate the predominant microbe. This isparticularly useful in the study of anaerobic bacteria which havefastidious culture requirements which make then difficult to culture.This is even more particularly useful for isolating and identifyinganaerobic bacteria from dental plaques which are difficult to cultureusing conventional procedures. DNA or RNA may be extracted, subjected toPCR by the universal primers and then the amplified fragment isolatedand sequenced and the organism identified by BLAST/GAP or other computeranalysis.

Reference herein to a “primer” or “probe” is not to be taken as anylimitation as to structure, size or function. The primer may be used asan amplification molecule or may be used as a probe for hybridizationpurposes. The preferred form of the molecule is as a primer foramplification.

Reference herein to a “nucleic acid primer” includes reference to asequence of deoxyribonucleotides or ribonucleotides comprising at least3 nucleotides. Generally, the nucleic acid primer comprises from about 3to about 100 nucleotides, preferably from about 5 to about 50nucleotides and even more preferably from about 5 to about 25nucleotides. A primer having less than 50 nucleotides may also bereferred to herein as an “oligonucleotide primer”. The primers of thepresent invention may be synthetically produced by, for example, thestepwise addition of nucleotides or may be fragments, parts, portions orextension products of other nucleotide acid molecules. The term “primer”is used in its most general sense to include any length of nucleotideswhich, when used for amplification purposes, can provide a free 3′hydroxyl group for the initiation of DNA synthesis by a DNA polymerase.DNA synthesis results in the extension of the primer to produce a primerextension product complementary to the nucleic acid strand to which theprimer has hybridized. The primer or probe may also be considered as atrapping or anchoring moiety from the target DNA or RNA.

The extension of the hybridized primer to produce an extension productis included herein by the term “amplification”. Amplification generallyoccurs in cycles of denaturation followed by primer hybridization andextension. The present invention encompasses from about 1 cycle to about120 cycles, preferably from about 2 to about 70 cycles and even morepreferably from about 5 to about 40 cycles including about 10, 15, 20,25 and 30 cycles.

In a particularly preferred embodiment, preparation of the sample isconducted in the presence of a nuclease inhibitor.

The assay may be conducted in any of a number of forms. In one example,an immobilized form of the assay is contemplated. In one embodiment, ageneric primer is immobilized to a solid support to capture targetDNA/RNA. Solution phase forward and reverse primers and the probe arethen used to perform the Real-Time PCR or by related or equivalenttechnology. In an alternative embodiment, one of the forward or reverseprimers is used as the capture molecule.

In accordance with this aspect of the present invention, a sample ofnucleic acid to be tested for the presence of bacteria is added to achamber, well or other receptacle comprising an immobilized nucleic acidcapture molecule. The capture molecules comprise a nucleotide sequencesubstantially complementary to a portion of either the target nucleotidesequence or a nucleotide sequence within a nucleic acid moleculecomprising the target sequence. The terms “captive molecule” and“primer” may be used interchangedly.

The capture molecule may be immobilized to the solid phase by anyconvenient means. The solid phase may be any structure having a surfacewhich can be derivatized to anchor a nucleic acid primer or othercapture molecule. Preferably, the solid phase is a planar material suchas the side of a microtitre well or the side of a dipstick.

The anchored nucleic acid molecule generally needs to be able to capturea target nucleic acid molecule by hybridization and optionallyparticipate in an amplification reaction. Alternatively, the anchorednucleic acid molecule will capture amplified nucleic acid molecules.

Methods for linking nucleic acid molecules to solid supports are wellknown in the art. Processes for linking the primer to the solid phaseinclude amide linkage, amidate linkage, thioether linkage and theintroduction of amino groups on to the solid phase. Examples of linkageto a solid phase can be found in International Patent Application No.PCT/AU92/00587 [WO 93/09250].

The anchored primer may also participate with one of the solution phaseprimers for amplification. Alternatively, a “generic” primer is anchoredto the solid support in order to amplify the nucleic acid moleculecomprising a target sequence. Specific amplification of the targetsequence can then be achieved by solution phase primers. In relation tothe latter embodiment, the solution would contain two solution phaseprimers and a labelled probe.

Anchored primers may also be used to trap target DNA or RNA forsubsequent cloning and/or sequencing (generally after amplification)and/or interrogation by probes or primers to identify a genus or speciesof microorganism or the predominant microorganism.

The method of the present invention provides an efficient, costeffective and accurate means of detecting particular nucleic acidmolecules and thereby quantitating bacterial load.

As stated above, the universal primers and probes of the presentinvention are also useful as a trap for total microbial-derived targetmaterial. Such trapped material may then be sequenced, or cloned andsequenced and/or subjected to primer/probe interrogation. Consequently,the present invention provides an ability to detect bacteria fromsamples which are difficult to cultivate and that would in allpracticality remain undetected or under-estimated by viable culturecount methods or, alternatively, bacteria that are in an aggregated orcoaggregated state or contaminated with matrix material, such as incarious dentine samples, where fluorescent detection and/or microscopicenumeration are also impractical. In addition, the application of theuniversal primers and probes of the present invention enable rapiddifferentiation of bacteria from viral infections within the limitedtime constraints sometimes experienced in life-threatening clinicalsituations. This is particularly useful, for example, in assessingencephalitis and distinguishing between microbial and viralencephalitis. In the field of clinical microbiology, the presentinvention enables the trapping and identification of, the predominatebacterium in an infection which leads to more efficacious treatmentprotocols. Any and all applications of the subject method areencompassed by the present invention.

The present invention is applicable to a range of industries includingthe medical, agricultural and industrial industries with specific usesincluding enviroprotection, bioremediation, medical diagnosis, waterquality control or food quality control.

Yet another aspect of the present invention is directed to a kit incompartmental form, said kit comprising a compartment adapted to containone or more primers capable of participating in an amplificationreaction of DNA comprising or associated with 16S rDNA or 16S rRNA,another compartment comprising a probe labelled at its 5′ end by afluorogenic reporter molecule and at its 3′ end by a molecule capable ofquenching said fluorogenic molecule and optionally another compartmentadapted to contain reagents to conduct an amplification reaction andoptionally a compartment adapted for extraction of nucleic acid fromcells.

In an alternative embodiment, the kit comprises a microtitre tray withtwo or more wells and with the reagents including the primers in thewells.

One or more of the primers may also be immobilized to the compartments.

The kit may conveniently be adapted for automated or semi-automated use.

The kit may also comprise a compartment for nucleic acid extraction.

The kit may also comprise an array of primers or probes to permitdetection of not only total Procarya but also other microorganisms orspecific bacteria.

The present invention further provides an extraction procedure forextracting nucleic acid material for amplification by the universalprimer/probe set.

Accordingly, the present invention contemplates a method for extractingnucleic acid material from a sample comprising microbial cells, saidmethod comprising subjecting a concentrated sample of said cells toenzymatic degradation and lysing said cells in the presence of SDS andthen purifying said nucleic acid material.

Preferably, the enzymatic treatment comprises treatment with aproteinase K and lysozyme and/or mutanolysin or their equivalents.Preferably, the lysed cells are also treated with an RNase.Conveniently, DNA or RNA is then specifically isolated.

This method is referred to as a single step DEPC method.

A two-step DEPC method is further contemplated by the present inventionand this could include a pressure-mediated cell lysis step (such as bysonication) or incubation on ice, in the presence of DEPC prior toenzymatic treatment.

Accordingly, the present invention further provides a method forextracting nucleic acid material from a sample comprising microbialcells, said method comprising subjecting a concentrated sample of saidcells to pressure-mediated disruption, or incubation on ice, in thepresence of DEPC prior to enzymatic degradation and then lysing saidcells in the presence of SDS and then purifying said nucleic acidmaterial.

Preferably, the pressure-mediated disruption is sonication. The otherpreferred aspects of this two-step method are the same as the one-stepmethod.

In a particular preferred embodiment, the one- or two-step extractionmethods are used in combination with the universal primers/probe set toenumerate and optionally identify particular bacteria in a sample.

Accordingly, the present invention contemplates a method for determiningmicroorganisms in a sample, said method comprising:

-   -   optionally subjecting a concentrated sample of said cells to        pressure-mediated disruption or incubation on ice, in the        presence of DEPC followed by enzymatic degradation and then        lysing said cells in the presence of SDS and then purifing said        nucleic acid material;    -   amplifying said nucleic acid material in the presence of forward        and reverse primers capable of hybridizing to a conserved        nucleotide sequence within 16S rDNA or 16S rRNA;    -   optionally detecting the presence of amplified product in the        presence of a probe labelled with a reporter molecule and        determining the total microbial content; and    -   optionally isolating the amplified product and either sequencing        the isolated product or subjecting the amplified product to        genetic interrogation to identify the genus or species of        microorganism present.

The present invention is further described by the following non-limitingExamples.

EXAMPLE 1 Bacterial Strains and Culture Conditions

Escherichia coli strains JM109, NM522 and XL 1 blue (Stratagene, LaJolla, Calif., USA) were available from previous studies. Staphylococcusaureus strains ATCC 12600, ATCC 9144, ATCC 12598, ATCC BM 10458 and ATCCBM 1014; Staphylococcus epidemidis strains ATCC 35983 and ATCC 14990;Staphylococcus hemolyticus ATCC 29970 and S. hemolyticus-infiltrativekeratitis isolate; Staphylococcus schleferi ATCC 43808; Pseudomonasaeurginosa strains ATCC 19660, ATCC 15442, ATCC 6294 and ATCC 6206;Pseudomonas fluorescens-infiltrative keratitis isolate; Pseudomonasputida-lens saline isolate; Pseudomonas stutzeri-infiltrate isolate;Pseudomonas alcaligens laboratory-isolate; Pseudomonas species andSerratia marcescens ATCC 274 were provided by the Co-operative ResearchCentre for Eye Research and Technology, The University of New SouthWales, Australia. All Escherichia, Staphylococcus, Pseudomonas andSerratia species were grown in Luria Burtanni broth at 37° C.Streptococcus mutans LT 11 and Streptococcus sanguis ATCC 10556 weregrown at 37° C. on Brain Heart Infusion broth (Oxoid, Basingstoke, UK)under 95% N₂/5% v/v CO₂ ; Fusobacterium nucleatum ATCC 25586,Fusobacterium necrophorum ATCC 25286, Actinomyces israelii ATCC 12102and Actinomyces naeslundii ATCC 12104 were obtained from the AmericanType Culture Collection (Rockville, Md., USA) and grown at 37° C. in aBrain Heart Infusion broth in an anaerobic chamber (85% v/v N₂, 5% v/vCO₂, 10% v/v H₂). Porphyromonas gingivalis ATCC 33277, Prevotellamelaninogenica ATCC 25845, Prevotella loescheii ATCC 15930,Peptostreptococcus micros ATCC 33270 and Peptostreptococcus anaerobiusATCC 27337 were obtained from the American Type Culture Collection(Rockville, Md., USA) and grown at 37° C. on CDC broth (1% v/vtrypticase peptone, DIFCO Becton Dickinson, Md., USA; 1% v/v trypticasesoy broth, DIFCO Becton Dickinson, Md., USA; 0.5% w/v sodium chloride,1% w/v yeast extract, Oxoid, Basingstoke, UK; 0.04% w/v L-cysteine,Sigma Chemical Co., St Louis, Mo., USA) containing 1% w/v hemin, 0.4%w/v menadione and 2% v/v horse serum in an anaerobic chamber (85% v/vN₂, 5% v/v CO₂, 10% v/v H₂). Porphyromonas endodontalis ATCC 35406American Type Culture Collection (Rockville, Md., USA) was also grown inan anaerobic chamber. Lactobacillus acidophilus ATCC 4356 andLactobacillus rhamnosus ATCC 7469 from the IDR culture collection weregrown at 37° C. in MRS broth (Oxoid, Basingstoke, UK) undermicroaerophilic conditions (95% v/v N₂, 5% v/v CO₂).

EXAMPLE 2 Source of Carious Dentine

Twenty carious teeth were obtained with informed consent from randomlyselected patients who presented with pain and requested extraction torelieve their symptoms. Patients were excluded from the study if theyreported a history of significant medical disease or anti-microbialtherapy within the previous four months. Unrestored teeth with coronalenamel and dentine caries were selected for inclusion in the study onthe basis of clinical diagnostic tests which indicated that they werevital, with clinical symptoms of reversible pulpitis (pain andheightened sensitivity to hot and cold stimuli).

Immediately after extraction, each tooth was placed in a container ofreduced transport fluid (RTF) (24) and transferred to an anaerobicchamber at 37° C. containing 85% N₂, 5% CO₂ and 10% H₂ v/v/v.Superficial plaque and debris overlying the carious lesion was removedand the surface rinsed several times with RTF. Using sterile sharpexcavators, all the softened and carious dentine was collected as smallfragments from each tooth. Sampling was completed within 20 min of toothextraction.

EXAMPLE 3 Determination of Colony-forming Units in Carious Dentine

The carious dentine extracted from each tooth was individually weighedand a standard suspension of 10 mg wet wt dentine (ml RTF)⁻¹ wasprepared at 37° C. in an anaerobic chamber (see Example 2). The dentinefragments were homogeneously dispersed in RTF by first vortexing for 20s and then by homogenizing by hand in a 2 ml glass homogenizer for 30 s.Samples (100 μl) of 10⁻³ to 10⁻⁶ serial dilutions of these suspensionswere prepared in RTF and plated in duplicate onto Trypticase Soy agar(Oxoid) containing 1 μg menadione ml⁻¹, 5 μg haemin ml⁻¹, 400 μgL-cysteine ml⁻¹ (Sigma) and 5% v/v horse blood (Amyl Media) (10). Theplates were incubated at 37° C. in an anaerobic chamber containing 85%N₂, 5% CO₂ and 10% H₂ v/v/v for 14 days and the number of colony-formingunits counted to determine the total microbial load (mg wet wt ofdentine)⁻¹. The unused dispersed carious dentine samples were frozen at−80° C.

EXAMPLE 4 Determination of Viable Bacteria from In Vitro Cultures

Viable cell counts of cultures of E. coli, P. aeruginosa and S. aureuswere determined by plating 100 μl of a 10⁻⁶ dilution of the appropriateculture grown in LB broth on LB agar plates and counting the coloniesafter aerobic incubation at 37° C. for 24 h.

EXAMPLE 5 Extraction of DNA from Bacterial Cultures

DNA was isolated from individual bacterial species using either theQIAamp DNA. Mini kit (QIAGEN, Clifton Hill, VIC) according to themanufacturer's instructions or using the freeze-boil method. In thelatter instance, bacterial cells from a 250 μl of culture were obtainedby centrifugation (14,000×g for 2 min at room temperature) andresuspended in 45 μl 10 mM phosphate buffer pH 6.7 prior to freezing at−20° C. The frozen cells were then heated in a boiling water bath for 10min.

EXAMPLE 6 Extraction of Anaerobic Bacterial DNA from Carious Dentine

Frozen suspensions of homogenized carious dentine were thawed on ice and80 μl samples removed and combined with 100 μl ATL buffer (Qiagen) and400 μg proteinase K (Qiagen). The samples were vortexed for 10 s andthen incubated at 56° C. for 40 min with periodic vortexing for 10 severy 10 min to allow complete lysis of the cells. This procedure wasfound to extract DNA from both Gram-negative and Gram-positive anaerobicbacteria in line with the finding that the cell-wall integrity ofGram-positive anaerobes is compromised when the bacteria are exposed tooxygen (11). Other micro-aerophilic or facultative Gram-positivebacteria including streptococci, lactobacilli and Actinomyces were notlysed by this procedure. Following the addition of 200 μg RNase (Sigma),the samples were incubated for a further 10 min at 37° C. DNA free ofcontaminating RNA was then purified using the QIAmp DNA Mini Kit(Qiagen) according to the manufacturer's instructions.

EXAMPLE 7 Sources of Other Bacterial DNA

DNA from Legionella pneumophila serogroup 4 ATCC 33156, serogroup 5 ATCC33216, serogroup 6 ATCC 33215, serogroup 1 Knoxyille-1 ATCC 33153,philadelphia-1 as well as Legionella anisa, Legionella bozenianiiserogroup-2, Legionella londineenisis, Legionella maceachernii andLegionella waltersii were provided by The Infectious DiseasesLaboratories, Institute of Medical and Veterinary Science, SouthAustralia; and those from Mycobacterium tuberculosis H37RV by TheMicrobiology Laboratory, Westmead Hospital, New South Wales, Australia.

EXAMPLE 8 DNA Sequence Analysis

The 16S rDNA sequences representing most of the Groups of bacteriaoutlined in Bergey's Manual (registered trade mark) of DeterminativeBacteriology (12) that were analyzed for construction of a universalprimers-probe set included (GenBank Accession Number in parentheses)Bacteroides forsythus (AB035460), P. gingivalis (POYRR16SC), P.melaninogenica (PVORR16SF), Cytophaga baltica (CBA5972), Campylobacterjejuni (CAJRRDAD), Helicobacter pylon (HPU00679), Treponema denticola(AF139203), T. pallidum (TRPRG16S), Leptothrix mobilis (LM16SRR),Thiomicrospira denitrificans (TDE243144), Neisseria meningitidis(AF059671), Actinobacillus actinomycetemcomitans (ACNRRNAJ), Haemophilusinfluenzae (HIDNA5483), E. coli (ECAT1177T), Salmonella typhi (STRNA16),Vibrio cholerae (VC16SRRNA), Coxiella burnetii (D89791), L. pneumophila(LP16SRNA), P. aeruginosa (PARN16S), Caulobacter vibrioides (CVI009957),Rhodospirillum rubrum (RR16S107R), Nitrobacter winogradskyi (NIT16SRA),Wolbachia species (WSP010275), Myxococcus xanthus (MXA233930),Corynebacterium diphtheriae (CD16SRDNA), M. tuberculosis (MTRRNOP),Streptomyces coelicolor (SC16SRNA), A. odontolyticus (AO16SRD), Bacillussubtilis (AB016721), S. aureus (SA16SRRN), Listeria monocytogenes(S55472), Enterococcus faecalis (AB012212), L. acidophilus (LBARR16SAZ),S. mutans (SM16SRNA), Clostridium botulinum (CBA16S), P. micros(PEP16SRR8), Veillonella dispar (VDRRNA16S), F. nucleatum (X55401),Chlamydia trachomatis (D89067) and Mycoplasma pneumoniae (AF132741). The16S rDNA sequences were aligned using the GCG program Pileup (22)accessed through the Australian National Genomic Information Service(ANGIS, http://www.angis.org.au). Regions of identity were assessedmanually for the design of the universal probe and primers (FIGS. 1A,1B, 1C) and then checked for possible cross hybridization with otherbacterial genes using the database similarity search program BLAST (23),also accessed through ANGIS. The Primer Express Software provided byApplied Biosystems to determine the appropriate primer/probecombinations was of limited value in this exercise and was only used tocheck for primer-dimer or internal hairpin configurations. Oncedesigned, the probe and primer sequences (Table 1) were synthesized byApplied Biosystems.

EXAMPLE 9 PCR Conditions

Amplification and detection of DNA by Real-Time PCR was performed withthe ABI-PRISM 7700 Sequence Detection System (PE Biosystems, FosterCity, Calif., USA) using optical grade 96 well plates. For determinationof the predominantly anaerobic Gram negative bacterial load in cariousdentine, the PCR reaction was carried out in triplicates in a totalvolume of 25 μl using either the TaqMan (registered trade mark) PCR CoreReagent Kit, PE Biosystems (Foster City, Calif., USA) to which was added200 μM of each dNTP, 3.5 mM MgCl₂, 0.625 U AmpliTaq Gold in 1×PCR buffersupplied by PE Biosystems (Foster City, Calif., USA) using 300 nMforward and reverse primers and 175 nM fluorogenic probe. Alternatively,the TaqMan (registered trade mark) Universal PCR Master Mix (PEBiosystems, Foster City, Calif., USA) was used containing 100 nM of eachof the universal forward and reverse primers and the fluorogenic probe.The reaction conditions for amplification of DNA were 50° C. for 2 min,95° C. for 10 min and 40 cycles of 95° C. for 15 s and 60° C. for 1 min.Data were analyzed using the Sequence Detection System Software from PEBiosystems (Foster City, Calif., USA) and are presented as the mean ofduplicate samples.

EXAMPLE 10 DNA Isolation Procedures

-   (i) Sonication: Bacterial cells pelleted at 14,000×g for 2 min at    room temperature were resuspended in 10 mM phosphate buffer pH 6.7    containing glass beads and were sonicated for 5 min, 10 min and 15    min, with 75 watts output using a Branson sonifier model 250.    Aliquots were collected at each time interval.-   (ii) Freeze-thaw method: The cell pellet was resuspended in 10 mM    phosphate buffer pH 6.7, frozen at −20° C., and after thawing, an    aliquot was used for the PCR reaction.-   (iii) Freeze-boil method: Bacterial cells pelleted at 14,000×g for 2    min at room temperature were resuspended in 10 mM phosphate buffer    pH 6.7, frozen at −20° C. and placed in boiling water for 10 min    before using for the PCR reaction.-   (iv) Enzymatic method: Bacterial cells pelleted at 14,000×g for 2    min at room temperature were resuspended in 10 mM phosphate buffer    pH 6.7 containing lysozyme and mutanolysin (each with 1 mg/ml final    concentration) and incubated at 60° C. for 30 min and lysed with SDS    (1% w/v final concentration).-   (v) QIAmp DNA Mini kit method: Total cell DNA was extracted from    bacterial cultures with the QIAmp DNA Mini kit (QIAGEN) as per the    manufacturer's instructions.-   (vi) ZnCl₂/EDTA/DEPC method: Bacterial cells' pelleted at 14,000×g    for 2 min at room temperature were resuspended in 10 mM phosphate    buffer pH 6.7 containing lysozyme and mutanolysin (each with 1    mg/ml-final concentration) and 5 mM ZnCl₂ or 100 mM EDTA or 20 mM    DEPC. After incubation at 60° C. for 30 min, the cells were lysed    with 1% w/v SDS (final concentration). DNA was purified from    bacterial cultures with the QIAmp DNA Mini kit as per the    manufacturer's instructions.

EXAMPLE 11 Protection from Nucleases

Purified preparation of DNA and P. gingivalis cell extract wereincubated at 60° C. for 30 min in the presence or absence of ZnCl₂ (5mM) or EDTA (100 mM) or DEPC (20 mM) or rabbit muscle actin (1 μg/ml) ordipyridyl (2 mM/5 mM), to assess their effect as nuclease inhibitors. Analiquot was checked on 1% w/v agarose gel electrophoresis.

EXAMPLE 12 Design of Universal Primers and Probe

Applied Biosystems has set a number of guidelines for the design ofprimers and probes. These include the fact that the melting temperature(T_(m)) of the DNA should be between 58-60° C. for the primers and68-70° C. for the probe; the G+C content should be between 30-80%; thereshould be no runs of more than three consecutive G's in either theprimers or the probe; there should be no more than two GC's in the lastfive nucleotides at the 3′ end of the primers; there should be no G onthe 5′ end of the probe; the selection of the probe should be from thestrand with more C's than G's and the amplicon length should be between50-150 bp.

The inventors then designed a set of universal primers and a probe basedon the sequence of 16S rDNA which would substantially comply with atleast most of the guidelines set by Applied Biosystems and also detect abroad range of bacterial species. In the inventors' hands, it was notpossible to meet all of these criteria. The inventors' final choice fora universal primers-probe set, however, only deviated in two ways fromthe ideal. These were the length of the amplicon and the number of GC'sin the last five nucleotides of the forward primer. The primers-probeset designed to act as a universal detection system for the Procarya byReal-Time PCR generated a 466 bp amplicon spanning residues 331 to 797on the E. coli 16S rRNA gene (Table 1). The selected probe and primersequences were highly conserved in all groups of Procarya (12) for whichrepresentative bacterial 16S rRNA genes were aligned (FIG. 1).

Although the multiple alignment of the selected bacterial 16S rRNAsequences show two mismatches in the forward primer of F. nucleatum(where the nucleotides are unknown) as well as a deletion in the 5′ endof the forward primer of P. micros, these discrepancies were toleratedduring Real-Time PCR since both genera could be quantified using theuniversal primers-probe set (Table 2).

To confirm the specificity for Procarya, the inventors searched a numberof available Eucarya and Archea databases available through ANGIS. TheBLAST search results showed only one significant hit—that of a specificbreast cancer cell line (BT029) detected only by the reverse primer.However, the human DNA sample supplied by Applied Biosystems in theirbeta-actin detection kit was not amplified by the primers-probe set andgave a totally negative result.

EXAMPLE 13 Sensitivity of the Universal Primers-probe Set in DetectingE. coli rDNA

TaqMan (registered trade mark) technology determines the PCR cycle atwhich the increase in fluorescence of the reporter dye reaches athreshold. This is known as the threshold cycle (C_(T)) and isproportional to the amount of target DNA and hence the number ofbacteria in the sample. The inventors produced a standard graph based onthe detection of E. coli rDNA, where one E. coli cell theoreticallyequates to the detection of 4.96 fg DNA (FIG. 2). Using E. coli as astandard, between 238 fg of E. coli DNA (corresponding to 48 E. colicells) and 2.38 ng of E. coli DNA (corresponding to 4.8×10⁵ E. colicells) was consistently detected. However, this does not take intoconsideration the number of rDNA copies on the E. coli genome. Thelimitation on the lower detection limit (i.e. between 4.8 cells to 48cells) varied with the use of the TaqMan (registered trade mark) PCRCore Reagent Kit or the TaqMan (registered trade mark) Universal PCRMaster Mix supplied by PE Biosystems (Foster City, Calif., USA). This isbelieved to be due to bacterial DNA contamination either in the enzymepreparation or in the chemical reagents used for PCR (13-16), anobservation verified in this study by detection using the universalprimer-probe set of rDNA in reagent mixes and negative controlscontaining no added E. coli DNA (FIG. 3). Although 40 PCR cycles areavailable with the universal primers-probe set, in theno-template-control, the fluorescent signal was consistently detectedaround a C_(T) of 33 and 38

EXAMPLE 14 Broad Range Detection and Relative Determination of BacterialNumber

In order to determine the relative total bacterial load for a givenspecies, the inventors compared the C_(T) value for the test sample witha standard graph derived from known amounts of E. coli DNA (FIG. 2). Thestandard graph was preferably prepared from data accumulated at the sametime as the test samples in order to act as an internal control. Byusing the standard curve, both the relative concentration of DNA in thesample and the relative number of bacteria could be determined for allselected species that represent the major Groups of bacteria listed inBergey's Manual (registered trade mark) of Determinative Bacteriology(12) [Table 2]. For each of these species, there was little variance inthe value of 2.00×10² (range 1.98-2.06×10²) bacteria per pg DNA when E.coli DNA was used as a standard. This indicated that the source of DNAwas not influencing the level of detection and that the primers-probeset was equally efficient in detecting the DNA irrespective of thespecies from which it was extracted. Similar conclusions could be drawnwhen different strains of the same species were detected by Real-TimePCR (Table 3).

EXAMPLE 15 Effect of the Source of Standard DNA on the measurement ofRelative DNA Concentration

Comparison with a DNA standard other than that of E. coli should resultin a difference in the relative amount of DNA detected due to variationsin rDNA copy number as well as the multiplying effect that thegeneration time (t_(d)) may have on this number. To confirm this, acomparison was made between the three rapidly growing aerobic bacteria,S. aureus, E. coli and P. aeruginosa, with t_(d) in vitro in the orderof 20-50 min and two slow growing obligate oral anaerobes, P.melaninogenica and P. endodontalis, with t_(d) in vitro in the order of5-15 h. The relative amount of DNA estimated by Real-Time PCR using eachof the 5 DNAs as standards was related to the amount of DNA determinedat A₂₆₀ nm (set at 100%). In each instance, it would be expected thatcomparison of like DNA by Real-Time PCR with the known amount of addedDNA would be approximately 100%. In two instances this was not the case.For both P. aeruginosa and P. melaminogenica approximately twice theamount of DNA was detected. This was due in part to the fact that therelative amounts of DNA were calculated by the Sequence Detection SystemVersion 1.6.3 software supplied by Applied Biosystems based upon thearbitrary placement of the horizontal threshold line used to determinethe C_(T) (as seen in FIG. 3). The horizontal threshold line wastherefore adjusted to bring these two values as close to 100% aspossible and the relative amount of DNA recalculated (Table 4).

As expected, variation in the relative amount of DNA was observed whenthe standard DNA differed from that of the species being evaluated(Table 4). However, significant error (>3-fold) was only observed whenthe fast growing aerobic bacteria were compared with the DNA standardsof the slow growing obligate anaerobes (over estimation) or conversely,when the obligate anaerobes were compared to the DNA of the fast growingaerobes (under estimation) (Table 4).

One of the values, that of the amount of S. aureus DNA detected usingthe P. melaninogenica DNA, was approximately two-fold greater thanexpected. However, this value was calculated from a low C_(T) valuewhere significant error can arise due to the logarithmic scale of theabscissa in the graph of C_(T) vs (DNA). At extreme high and low C_(T)values, a two-fold error in the estimation of the relative amount of DNAcan occur. By taking this inherent two-fold error into account and bysubsequently altering one of the 25 values for the relative amount ofDNA by a factor of two (Table 4—see footnote ‡), the data in Table 4allowed an estimation of the ratio of the number of copies of the 16SrRNA operons in the different species. An average ratio of 23:13:10:2:1(to the nearest integer) for the copy numbers in S. aureus, E. coli, P.aeruginosa, P. endodontalis and P. melaninogenica respectively fittedthe modified data. This implied that the fast growing aerobes, S.aureus, E. coli and P. aeruginosa possessed approximately twice theknown chromosomal complement of 16S rRNA operons. The data alsopredicted that the obligate anaerobes possess only one or two 16S rRNAoperons per chromosome. The exact copy numbers are currently unknown.

EXAMPLE 16 Comparison of Viable Cell Numbers and the Relative Estimationof Bacteria in an Artificial In Vitro Mixture using Real-Time PCR

In order to determine the validity of using the universal primers-probeset to estimate the total number of bacteria in a mixed culture, thethree bacteria, E. coli, P. aeruginosa and S. aureus, were grownseparately in vitro to stationary phase and equal volumes of the threecultures (2 ml) mixed together. The number of E. coli, P. aeruginosa andS. aureus colony forming units at stationary phase were determined byserial dilution on agar plates and compared with the relative bacterialload determined by Real-Time PCR using the universal primers-probe setand E. coli DNA as the standard. A consensus was noted in the estimationof bacterial counts irrespective of the method used (Table 5), despitethe fact that the number of copies of the 16S rRNA operons in a singlechromosome of E. coli is 7 while that in P. aeruginosa is 4 and S.aureus is 9, and the expectation that P. aeruginosa would beunder-estimated and S. aureus over-estimated against the E. colistandard DNA.

EXAMPLE 17 Comparison of the Number of Anaerobic Bacteria in CariousDentine by Real-Time PCR with the Total Anaerobic Colony Count

The value of using the universal probe and primers set in estimating theanaerobic bacterial load in carious dentine was determined in twentyclinical samples using P. melaninogenica ATCC 25845 DNA fromanaerobically grown cells as the standard. Comparison was made with thetotal anaerobic colony count for each of the samples. The mean number ofanaerobic bacteria determined by Real-Time PCR was 3.6×10⁸ (mgdentine)⁻¹ (range 1.1×10⁸-1.1×10⁹ [mg dentine]⁻¹), while that for thetotal viable cell count was 1.1×10⁷ (mg dentine)⁻¹ (range2.0×10⁶-3.7×10⁷ [mg dentine]⁻¹). The results indicated that theculture-based technique under-estimated the total bacterial load incarious dentine, since the number of anaerobic bacteria that weredetected in the samples by Real-Time PCR was, on average, 40-foldgreater than that detected by colony counts despite the fact that thelatter also contained facultative Gram-positive bacteria (Table 6).

EXAMPLE 18 Sonication of Bacterial Cells for Isolation of Bacterial DNA

To eliminate loss of DNA using a multistep sample preparation protocol,bacterial cell suspensions were sonicated to release DNA from cells forquantification using Real-Time PCR. DNA was released more effectivelywhen the cells were sonicated using glass beads. Sonicates of S. mutansand P. gingivalis were diluted to the appropriate concentration andchecked in the ABI-PRISM 7700 Sequence Detection System forquantification of DNA using the universal primers-probe set. The effectof sonication was compared with DNA isolation using freeze-thaw orfreeze-boil. As seen in FIG. 4, the freeze-boil technique methodreleased most DNA. Increased sonication times had little effect on DNArecovery from S. mutans, but had a negative effect on P. gingivalisrecovery

EXAMPLE 19 Presence of Nucleases in P. Gingivalis as Seen on Agarose GelElectrophoresis

The presence of nucleases in P. gingivalis was checked using 1% w/vagarose gel electrophoresis. Exogenous, purified, P. gingivalis DNA wasincubated at 50° C. for 30 min with each of the DNA isolation fractionsshown in FIG. 5A and when loaded on a 1% w/v agarose gel, intact DNAcould be detected only after boiling the frozen culture, as seen in FIG.5A.

EXAMPLE 20 The Critical Role of Nucleases and the Effect of ZnCl₂ on theQuantification in Individual and Mixed Bacterial Populations

DNA isolated from P. gingivalis, in the absence or presence of E. colior S. mutans was checked in the ABI-PRISM 7700 Sequence DetectionSystem, for quantification of DNA, using the universal primers-probe setand appropriate dilution of the sample. A significant increase inquantification of DNA was evident in individual and mixed bacterialpopulations in the presence of 5 mM ZnCl₂ (FIGS. 6 a, 6 b).

EXAMPLE 21 Effect of Removal of ZnCl₂ and SDS on Quantification UsingUndiluted Samples

To eliminate the interference of ZnCl₂ and SDS in undiluted or lowerdilution samples, it was necessary to remove the nuclease inhibitor andcell lysis agent, respectively, before the DNA samples were analyzed inthe ABI-PRISM 7700 Sequence Detection System. A P. gingivalis cellpellet, resuspended in 10 mM phosphate buffer pH 6.7, containinglysozyme and mutanolysin (each with 1 mg/ml-final concentration) and 5mM ZnCl₂ was incubated at 60° C. for 30 min and then lysed with 1% w/vSDS, before purification of DNA using the QIAamp DNA Mini kit.Quantification of DNA could not be done in undiluted samples. This waspossibly due to high concentrations of ZnCl₂ in the undiluted samplesthat could interfere with the PCR reaction. Purification of DNA usingthe QIAamp Mini kit restored the amount of DNA quantified as seen inFIG. 7.

EXAMPLE 22 Internal Positive Control Using B. tryoni dsX Gene Insert inpGEM (Registered Trade Mark)-T Easy Vector System

A TaqMan (registered trade mark) exogenous, internal positive controlwas designed to be used with the ABI PRISM 7700 Sequence DetectionSystem to determine the efficiency of DNA recovery following samplepreparation and to evaluate the effect of any PCR inhibitors in thereaction. The forward primer 5′GGAAGGTAAGTTGCATTTCAGCA3′ [SEQ ID NO: 4],reverse primer 5′GCGTACTTATCATGGTAAATTAAGTCAATT3′ [SEQ ID NO:5] andfluorogenic probe, VIC-TCCCGTTACAAAATCGTGTTTACATCGTATACTCG [SEQ ID NO:6]were designed from the reported sequence of the dsX gene of Bactrocerratryoni (GenBank Accession No. AF040077) using Primer Express software(Applied Biosystems, Foster City, Calif., USA). The probe sequence forthis Internal Positive Control (IPC-BT-PG) was labelled with thefluorescent dye VIC at the 5′ end to differentiate the IPC from thespecies specific and universal probes which are labelled with thefluorescent dye FAM at the 5′ end.

B. tryoni dsX gene insert in pGEM (registered trade mark)-T Easy wasconfirmed by PCR and generated an 89 bp amplicon as seen on 2% w/vagarose gel electrophoresis. The chimeric plasmid also gave afluorescent signal in the ABI-PRISM 7700 Sequence Detection System,confirming an internal site of the probe in the amplicon (FIG. 8).

EXAMPLE 23 Isolation of P. gingivalis DNA in the presence of theinternal positive control

The P. gingivalis cell pellet (from 250 μl culture, spun at 14,000×g for2 min at room temperature) was resuspended in 10 mM phosphate buffer pH6.7 containing lysozyme and mutanolysin (each with 1 mg/ml-finalconcentration), 5 mM ZnCl₂ and the internal positive control (B. tryonidsX gene insert in pGEM (registered trade mark)-T Easy Vector System)and was incubated at 60° C. for 30 min and then lysed with 1% w/v SDS.The same amount of culture pellet was also resuspended in 10 mMphosphate buffer pH 6.7 containing the internal positive control andkept frozen at −20° C. The frozen sample was boiled for 10 min. Afterdiluting the sample, an aliquot was checked in the ABI-PRISM 7700Sequence Detection System. A higher amount of DNA (lower C_(T) value)was estimated for P. gingivalis and the internal positive control whenthe samples were either boiled or isolated in phosphate buffercontaining 5 mM ZnCl₂ (as seen in Table 7), whereas P. gingivalis DNAand the internal positive control were degraded (higher C_(T) value)when the nucleases were active in the freeze-thawed sample or in 10 mMphosphate buffer. The internal positive control could, therefore, beused to determine the efficacy of DNA recovery following samplepreparation.

EXAMPLE 24 Validation of Real-Time PCR Estimation of Porphyromonasgingivalis in Periodontal Plaque Sample by Sequence Based Identification

Using Real-Time PCR, contribution of Porphyromonas gingivalis incomparison to the total bacterial load in a diseased site periodontalplaque sample was estimated with P. gingivalis specific and universalprimers-probe set.

The inventors used a single Universal primer pair to amplify 466 bpfragment of DNA from the DNA isolated from diseased site humanperiodontal plaque sample. The primers and probes used are in Table 1.Of the 57 clones analyzed, Porphyromonas gingivalis, Bacteroidesforsythus, Prevotella tannerae, Rothia dentocariosa were identified tospecies level, where as Prevotella, Fusobacteria, Catonella, Clostridia,Desulfobubus, Cainpylobacter, Capnocytophaga and Treponema could beidentified to genus level. Predominance of P. gingivalis (29.8%) alongwith Fusobacteria (31.6%) followed by B. forsythlus (10.5%), Prevotella(7%) and Treponema (3.5%) is evident in Sequence based identification(Table 14A). All the other species were represented as one clone per 57clones analyzed. DNA isolated from same plaque sample was analyzed usingReal-Time PCR technology to estimate P. gingivalis number (using P.gingivalis primers-probe set, SEQ ID NOS:7, 8 and 9) in comparison tothe total load (using Universal primers-probe set). P. gingivalis cells(1.4×10¹¹) against total load (4.8×10¹¹) in this diseased site plaquesample showed that P. gingivalis represented 29% of the total load(Table 14B). This example shows the value of the universal primers totrap microbial 16S rDNA for subsequent analysis by sequencing. Thesedata very closely match with the Sequence based identification andvalidated the two results. Therefore, use of Real-Time PCR technology toestimate the load of P. gingivalis in periodontal plaque sample greatlyassists in clinical treatment modality.

EXAMPLE 25 Inhibition of Nuclease Activity and Removal of PCR InhibitorsImproves Efficiency of Quantifying Bacteria by Real-Time PCR

Methods for extracting and stabilizing DNA from representatives of amixed oral flora and comprising the microaerophilic Gram positiveorganisms, Streptococcus mutans, Lactobacillus acidophilus andActinomyces israelii, the Gram positive anaerobe Peptostreptococcusmicros, and the Gram negative anaerobes, Fusobacterium nucleatum,Porphyromonas endodontalis, Porphyromonas gingivalis and Prevotellamelaminogenica, were evaluated for quantitation using Real-Time PCR.

While DNA was easily extracted from the Gram negative organisms and theanaerobic P. micros, microaerophilic Gram positive species requireddigestion at 56° C. with a combination of lysozyme, mutanolysin andproteinase K. It was noted that P. gingivalis released potent broadspectrum DNAase activity that produced extensive degradation of DNA fromall of the test species as well as from an internal positive controlderived from the fruit fly B. tyroni. Inhibitors of DNAses weredifferentially effective and variably inhibitory to the hydrolasesnecessary for DNA release from Gram positive organisms. A consensusmethod for this disparate group of organisms was to pre-treat with thenuclease inhibitor diethyl pyrocarbonate (DEPC), digest with hydrolasesand add sodium dodecyl sulfate (SDS) to release DNA from the Gramnegative and Gram positive organisms. Subsequent purification of the DNAto remove the added DEPC and SDS and other potential PCR inhibitors wasalso necessary to accurately quantify the DNA, and hence the number ofbacteria in a sample. The efficiency of DNA recovery following samplepreparation was assessed by including a known amount of exogenous DNA(from B. tyroni) in the sample to act as an internal positive control.This standard also provides a control for other combinations ofmicroorganisms where unrecognised nuclease activities may be resistantto DEPC.

The following methods and materials were employed.

(i) Construction of an Internal Positive Control for Real-Time PCR

A chimeric plasmid was constructed to act as an internal positivecontrol. The portion of DNA in the chimeric plasmid that was detected byReal-Time PCR originated from the Queensland fruit fly, Bactrocerratryoni which was obtained from frozen (−80° C.) stocks at the Fruit FlyResearch Center, University of Sydney, NSW, Australia. Genomic DNA wasextracted from 30 flies (17) and the region between nucleotides 37 and126 of the dsX gene (GenBank Accession No. AF040077) amplified by PCR(FTS-320 Thermal Sequencer, Corbett Research, NSW, Australia) using 4 μgB. tryoni DNA, 100 nM of each of the forward and reverse primersdesigned for Real-Time PCR detection of this segment of DNA (Table 1),200 μM of each deoxyribonucleotide triphosphates, 3.5 mM MgCl₂ and 2.5 UAmpliTaq Gold in 1×PCR buffer (Applied Biosystems). The PCR reaction wascarried out in a volume of 50 μl at 95° C. for 10 min followed by 40cycles at 95° C. for 15 s and 60° C. for 1 min. The PCR amplicon (89 bp)from the entire 50 μl reaction volume was purified using the Wizard(registered trade mark) PCR Preps DNA Purification System (PromegaCorporation, Madison, Wis.). The purified PCR product was cloned intopGEM (registered trade mark)-T Easy Vector (Promega Corporation)according to the manufacturer's instructions. Competent E. coli XL blue1was transformed by electroporation (2.45 V) with the chimeric plasmidusing a Bio-Rad Gene Pulser. Recombinants were selected on LB agarplates containing 100 μg ampicillin per ml, 1 mMisopropy-β-D-lthiogalactoside and 100 μg5-bromo-4-cholro-3-indolyl-β-D-galactoside (X-Gal) per ml. The chimericplasmids carrying the 89 bp PCR amplicon for the dsX gene were isolatedusing the Wizard (registered trade mark) Plus SV Minipreps DNAPurification System (Promega Corporation) and termed IPC-BT.

(ii) Design of Primers-probe Sets

For the species specific quantification of Porphyromonas gingivalis, aprimers-probe set was designed from the 16S rDNA database accessedthrough the Australian National Genomic Information Service (ANGIS,http://www.angis.org.au). The P. gingivalis species specificprimers-probe set (SEQ ID NOS:7 and 8) (Table 1) generated a 150 bpamplicon spanning nucleotides 589 to 739 in the P. gingivalis 16S rDNAsequence (GenBank Accession No. L16492) with an internal site for thedual labelled fluorogenic probe (SEQ ID NO:9). The primers-probe setfulfilled the recommended guidelines set by Applied Biosystems (FosterCity, Calif.).

The design of a universal primers-probe set forth above. The universalprimers-probe set (Table 1) generated a 466 bp amplicon spanningresidues 331 to 797 on the E. coli 16S rRNA gene (GenBank Accession No.ECAT1177T) with an internal site for the dual-labelled fluorogenicprobe.

A primers-probe set was also designed to enable the detection of theexogenously added internal positive control, IPC-BT. The primers-probeset (Table 1) was designed from the sequence of dsX gene of B. tryoniusing Primer Express software (Applied Biosystems). The primers-probeset amplified a 89 bp region spanning nucleotides 37 to 126 on the dsXgene. The probe sequence for the IPC-BT was labelled with the reporterfluorescent dye VIC at the 5′ end to differentiate it from the speciesspecific and universal probes which were labelled at the 5′ end with thereporter fluorescent dye FAM (Table 1).

(iii) DNA Isolation Procedures

Different methods for releasing DNA by lysing bacteria were assessed.These included:

(a) Sonication:

-   -   P. gingivalis or S. mutans (˜10⁹ cells) were harvested by        centrifugation (14,000 g, 2 min, 18-20° C.) and resuspended in        200 μl of 10 mM phosphate buffer pH 6.7 containing 50 mg of        glass beads (0.10-0.11 mm diameter) prior to sonication for 5,        10 or 15 min at 75 W using a Branson Sonifier (model 250;        Branson Ultrasonics Corporation, Danbury, Conn.). Aliquots (50        μl) collected at each time interval and diluted 1000-fold were        used for Real-Time PCR. Quantification of DNA made use of the        universal primers-probe set (Table 1) and was based on a        standard graph generated by known amounts of E. coli DNA as        previously described.        (b) Freeze-thaw Method:    -   P. gingivalis or S. mutans (˜10⁹ cells) were harvested by        centrifugation (14,000 g, 2 min, 18-20° C.), and resuspended in        200 μl of 10 mM phosphate buffer pH 6.7 and frozen at −20° C.        After thawing, the sample was diluted 100 fold and a 5 μl        aliquot used for Real-Time PCR. Quantification of DNA made use        of the universal primers-probe set as described in (a) above.        (c). Freeze-boil Method:    -   P. gingivalis or S. mutans cells (˜10⁹ cells) were harvested,        resuspended and frozen at −20° C. (2-16 h) as described above        before being boiled for 10 min. After cooling to room        temperature (18-20° C.), samples were diluted 100-fold and 5 μl        aliquots used for Real-Time PCR using the universal primers        probe set as described in (a).        (d) Enzymatic Method:    -   P. gingivalis alone or mixed with either S. mutans or E. coli        (˜2.5×10⁸ of each bacterial species) were harvested by        centrifugation (14,000 g, 2 min, 18-20° C.) and resuspended in        either 45 μl (for P. gingivalis cells alone) or 90 μl (for P.        gingivalis in combination with S. mutans or E. coli cells) of 10        mM phosphate buffer pH 6.7 containing 1 mg lysozyme ml⁻¹ and 1        mg mutanolysin ml⁻¹. After incubation at 60° C. for 30 min, the        bacteria were lysed in the presence of 1% w/v SDS, before being        diluted 100-fold and 5 μl aliquots being used for Real-Time PCR.        Quantification of DNA made use of the universal primers-probe        set as described in (a).        (e) ZuCl₂ Method    -   P. gingivalis alone or mixed with either S. mutans or E. coli        (˜2.5×10⁸ of each bacterial species) were harvested and        resuspended as described in (d) above in the presence of 5 mM        ZnCl₂. After incubation at 60° C. for 30 min the cells were        lysed in the presence of 1% w/v SDS, before being diluted        100-fold and 5 μl aliquots being used for Real-Time PCR.        Quantification of DNA made use of the universal primers-probe        set as described in (a).        (f) Isolation of DNA using ATL Buffer From QIAamp DNA Mini Kit:    -   Bacterial cultures (˜5×10⁸ of each bacterial species) were        pelleted at 13000×rpm at 5 min in Bifuge pico (Heraeus). Cell        pellets were resuspended in 180 μl ATL buffer (Qiagen) and 400        μg proteinaseK (Qiagen). The cell suspensions were incubated at        56° C. for 40 min with intermittent vortexing for 10 s after        every 10 min. RNase (200 gg) was added, followed by further        incubation at 37° C. for 10 min. DNA was purified using QIAamp        DNA Mini Kit (Qiagen) as per the manufacturer's instructions.        (g) Isolation of DNA by One Step DEPC Method:    -   Bacterial cultures (˜5×10⁸ of each bacterial species) were        pelleted at 13000×rpm at 5 min in a Bifuge pico (Heraeus). The        cell pellet was resuspended in 200 μl buffer containing 10 mM        sodium phosphate pH 6.7, 20 mM DEPC, lysozyme (5 mg per ml        [final conc.]), mutanolysin (1000 U per 0.48 mg per mil [final        conc.]) and 400 μg proteinaseK (Qiagen). The cell suspensions        were incubated at 56° C. for 40 min with intermittent vortexing        for 10 s after every 10 min. Cells were lysed with SDS (1% w/v        [final conc.]). RNase (200 μg) was added, followed by further        incubation at 37° C. for 10 min. DNA was purified using a QIAmp        DNA Mini Kit (Qiagen) as per the manufacturer's instructions.        (h) Isolation of DNA in Mixed Bacterial Cultures by One Step        DEPC Method:    -   Bacterial cultures (˜2.5×10⁸ of each bacterial species) were        pelleted at 13000×rpm at 5 min in a Bifuge pico (Heraeus). Cell        pellets were resuspended in 200 μl buffer containing 10 mM        sodium phosphate pH 6.7, 20 mM DEPC, lysozyme (5 mg protein per        ml [final conc.]), mutanolysin (1000 U per 0.48 mg protein per        ml [final conc.]), 400 μg proteinaseK (Qiagen). The cell        suspensions were incubated at 56° C. for 40 min with        intermittent vortexing for 10 s after every 10 min. Cells were        lysed with SDS (1% w/v [final conc.]). RNase (200 μg) was added,        followed by further incubation at 37° C. for 10 min. DNA was        purified using a QIAmp DNA Mini Kit (Qiagen) as per the        manufacturer's instructions.        (i) Isolation of DNA by Two Step DEPC Method:    -   Bacterial cultures (˜5×10⁸ of each bacterial species) were        pelleted at 13000×rpm at 5 min in a Bifuge pico (Heraeus). Cell        pellets were resuspended in 144 μl buffer (10 mM sodium        phosphate pH 6.7) containing 27.8 mM DEPC. Cell suspensions were        incubated on ice for 10 min or sonicated in pulse or continuous        mode for 6 min at 75 W using a Branson Sonifier (model 250;        Branson Ultrasonics Corporation, Danbury, Conn.) followed by        addition of 56 μl of enzyme mix.: [lysozyme (5 mg protein per ml        [final conc.]), mutanolysin (1000 U per 0.48 mg protein per ml        [final conc.]), containing and 400 μg proteinaseK (Qiagen)]. The        cell suspensions were incubated at 56° C. for 40 min with        intermittent vortexing for 10 s after every 10 min. Cells were        lysed with SDS (1% w/v [final cone.]). RNase (200 μg) was added,        followed by further incubation at 37° C. for 10 min. DNA was        purified using a QIAmp DNA Mini Kit (Qiagen) as per the        manufacturer's instructions.        (iv) Detection of Nuclease Activity

Exogenous, P. gingivalis DNA (300-400 ng), purified using QIAmp DNA MiniKits (see (f) above), was added to samples containing 300-400 ng DNAprepared by lysing bacteria according each of the procedures describedin (a)-(c) above prior to incubation at 50° C. for 30 min. Exogenous DNAfrom Fusobacterium nucleatum, Porphyromonas endodontalis, Porphyromonasgingivalis, Prevotella melaninogenica and Peptostreptococcus micros andEscherichia coli (prepared using ATL buffer and QIAmp DNA Mini Kit) andStreptococcus mutans (prepared using one-step DEPC method) wereincubated at 50° C. for 30 min with P. gingivalis freeze-thaw extract(procedure described in (b)). The degree of DNA degradation wasdetermined visually following electrophoresis of samples on 1% w/vagarose gels.

(v) ZnCl₂ as a PCR Inhibitor

In order to determine whether ZnCl₂ acted as an inhibitor of Real-TimePCR, DNA was extracted from two sets of duplicate samples of P.gingivalis (˜5×10⁸ and ˜5×10⁷ cells) using the protocol described in (e)above. One of each set of duplicate samples of DNA was purified using aQIAmp DNA Mini Kits (QIAGEN). All samples were subsequently diluted totheoretically contain the same amount of DNA before subjecting toanalysis by Real-Time PCR using the universal primers-probe set.

(vi) Isolation of P. Gingivalis DNA in the Presence of the InternalPositive Control

DNA was extracted from P. gingivalis (2.5×10⁸ cells) in the presence of1 μl IPC-BT, using the protocol described in (e) above. Followingappropriate dilution, the amount of P. gingivalis DNA and IPC-BT DNAwere determined using the specific P. gingivalis and IPC-BTprimers-probe set, respectively.

(vii) Conditions for Real-Time PCR

Amplification and detection of DNA by Real-Time PCR made use of the ABIPRISM 7700 Sequence Detection System (Applied Biosystems, Foster City,Calif.) using a 96 well plate format. The PCR was carried out induplicate, in a 25 μl reaction volume containing 300 nM of each of theUniversal primers and 100 nM of the Universal probe or 100 nM of each ofthe primers and probe for the Internal Positive Control (Table 1) usingthe TaqMan (registered trade mark) PCR Core Reagents Kit (AppliedBiosystems). The reaction conditions for amplification of DNA were 95°C. for 10 min, and 40 cycles of 95° C. for 15 s and 60° C. for 1 min.Data was analyzed using the Sequence Detection Software Version 1.6.3supplied by Applied Biosystems.

(viii) Viable Count

P. gingivalis was grown in CDC broth under anaerobic conditions at 37°C. for four days and S. mutans was grown 16-18 h in BHI broth at 37° C.under 5% CO₂ . P. gingivalis culture (100 μl), diluted in CDC broth to10⁻⁶ dilution was plated on CDC agar and incubated under anaerobicconditions at 37° C. for four days and colonies were counted. S. mutansculture (100 μl), diluted in BHI broth to 10⁻⁶ dilution was plated onBHI agar and incubated under 5% CO₂ at 37° C. for 16-18 h and colonieswere counted.

The following results were obtained.

(I) Preparation of Bacterial Cells for the Isolation of DNA

To access all bacterial DNA, the bacterial cell suspensions weresonicated to release DNA for quantification using Real-Time PCR. DNA wasreleased more effectively when the cells were sonicated using glassbeads. Effect of sonication was compared with DNA isolation usingfreeze-thaw and freeze-boil methods. Freeze-thaw method released theleast DNA from P. gingivalis cells as well as S. mutans cells, whereasfreeze-boil method released most DNA from P. gingivalis cells ratherthan S. mutans cells. This indicated that boiling the samples could beeffective method for release of DNA from Gram negative bacteria but notGram positive bacteria. On the contrary, increase in the sonication timefrom 5 to 15 minutes, had detrimental effect on the quantification of P.gingivalis DNA with no significant change in the quantification of S.mutans DNA.

(II) Presence of Nucleases in P. Gingivalis

Agarose gel electrophoresis (1% w/v) confirmed the presence of nucleasesin P. gingivalis. Exogenous P. gingivalis DNA was completely degradedand could not be seen when incubated at 50° C. for 30 min in thepresence of freeze-thawed P. gingivalis culture. However, under the sameconditions intact DNA was detected after boiling the frozen P.gingivalis culture. Degradation of exogenous P. gingivalis DNA in thepresence freeze-thawed P. gingivalis culture could be prevented byaddition of 5 mM ZnCl₂ before incubating the samples at 50° C. for 30min. Exogenous DNA from Fusobacterium nucleatum, Porphyromonasendodontalis, Porphyromonas gingivalis, Prevotella melaninogenica andPeptostreptococcus micros and Streptococcus mutans was completelydegraded in the presence of freeze-thawed P. gingivalis culture (FIG.5B).

(III) Protection Against Nuclease Degradation by 5 mM ZnCl₂ using theABI—PRISM Sequence Detection System (ABI-SDS).

DNA isolated from P. gingivalis cells in the presence of E. coli cellsor S. mutans cells was quantified on the ABI-SDS using the universalprimers-probe set. Significant increase in the amount of DNA quantifiedwas evident for the individual and mixed bacterial populations when thesamples were prepared in the presence of 5 mM ZnCl₂.

(IV) Effect of ZnCl₂ as a PCR Inhibitor

When DNA was isolated in the presence of 5 mM ZnCl₂ and diluted 100 foldbefore using 5 μl on ABI-SDS, ZnCl₂ did not inhibit the PCR reaction. Asseen in the results for the neat culture, a final concentration of ZnCl₂in the PCR reaction to 0.005 mM caused minimal interference with theamplification reaction and there was no significant change in the amountof DNA quantified before and after the use of the QIAmp DNA Mini Kit.However, dilution of DNA 10 fold (as in the case of 10 fold dilutedculture) before using 5 μl on ABI-SDS, resulting in a finalconcentration of 0.05 mM ZnCl₂ in the PCR reaction, prevented theamplification of P. gingivalis DNA.

(V) The Internal Positive Control (IPC-BT)

The addition of a chimeric plasmid containing unique non-bacterial DNAto mixed bacteria samples allowed both the determination of theefficiency of DNA recovery following sample preparation and thedetection of potential PCR inhibitors in the reaction mix duringReal-Time PCR. B. tryoni dsX gene insert in pGEM (registered trademark)-T Easy was confirmed by PCR which generated an 89 bp ampliconvisualized on 2% w/v agarose gel electrophoresis.

(VI) Isolation of P. gingivalis DNA in the Presence of IPC-BT

Due to limitation of the software, the standard graph generated by FAMlabeled probes (P. gingivalis or universal) could not be used toquantify IPC-BT, as the reporter dye on the probe for detection ofIPC-BT is VIC labeled. This necessitated the results to be expressed interms of C_(T) values. Isolation of P. gingivalis DNA in the presence ofthe Internal Positive Control and the effect of nucleases on thequantification (expressed as C_(T) values) is shown in Table 8. P.gingivalis DNA and IPC-BT were degraded at the same time by the actionof the bacterial nucleases present in the sample when DNA was isolatedby freeze-thaw method or in the absence of ZnCl₂ (higher C_(T) value).On the contrary, isolation of DNA by the freeze-boil method or ZnCl₂method protected against degradation of DNA by the nucleases (lowerC_(T) value). Multiplexing the same samples showed no significantvariation on the levels of P. gingivalis DNA and IPC-BT in terms ofC_(T) values (Table 8).

(VII) Isolation of DNA using ATL Buffer Front QIAamp DNA Mini Kit

ATL buffer from the QIAamp DNA Mini kit could recover DNA from the Gramnegative bacteria Fusobacterium nucleatum, Porphyromonas endodontalis,Porphyromonas gingivalis, Prevotella melaminogenica and the anaerobicGram positive bacterium, Peptostreptococcus micros. However, DNArecovery from Streptococcus mutans, Actinomyces israeli andLactobacillus acidophilus was almost negligible (Table 9).

(VIII) Isolation of DNA by One Step DEPC Method

As can be seen (Table 10), in the absence of DEPC, Porphyromonasgingivalis DNA is significantly degraded. Recovery of DNA fromStreptococcus mutans improved more than 10-fold due to the cell walltreatment. However, the amount of DNA recovered from Peptostreptococcusmicros dropped by about 5-fold in the presence of DEPC. DNA recoveryfrom the remaining bacteria in this group remained comparativelyunaffected.

(IX) Comparison of Viable Count of P. gingivalis and S. mutans CellsBased on Isolation of DNA by One Step DEPC Method

Efficiency of DNA recovery by the ATL method and one-step DEPC methodand the number of P. gingivalis cells calculated based on these valueswere comparable. However, the viable count was 10-fold less than therelative number of cells estimated based on Real-Time PCR. For S. mutansthe number of viable cells per ml were comparable with the number ofcells per ml estimated, based on Real-Time PCR (Table 11).

(X) Isolation of DNA in Mixed Bacterial Cultures by One Step DEPC Method

In the absence of DEPC, the mixed culture reported lower recovery of DNAas compared to the presence of DEPC during DNA isolation (Table 12).(XI) Isolation of DNA by Two Step DEPC Method

Incubation of bacterial suspensions in the presence of DEPC prior tocell wall treatment enzymes improved the recovery of DNA fromPeptostreptococcus micros (compare data in Table 10 with that in Table13). Sonication for a 6 min. pulse (rather than continuous sonication)improved the recovery of A. israelii DNA by 3-fold and the amount of DNArecovered from all the other bacteria was comparable (compare data inTable 10 with that in Table 13).

EXAMPLE 26 Sequence Based Identification of Bacteria from Dental PlaqueFlora

The present method involves culturing bacteria from dental plaques anddetermining that they could not be readily identified by standardculture techniques. DNA is isolated by the two-step DEPC method andsubjected to PCR using the universal primers. The amplified product ispurified and sequenced and subjected to BLAST/GAP analysis.

Specifically, DNA was isolated from bacterial cultures using two-stepDEPC method. PCR reaction was run using universal primer set. Amplifiedproduct 466 bp was purified and sequenced using universal forwardprimer. DNA sequence (431 bp for 4-2, 400 bp for 2-2-1 and 1-2-1, 386 bpfor 6-5 and 10-34 and 382 bp for 4-2-1) was BLAST searched using NRnucleic database through WebANGIS. High score bacterial sequences weresubjected to GAP program to ascertain % similarity and % identity.Identification of the culture was based on more than 98.5-99% identicalsequences (as specified with identification number) using the ampliconlength for each culture as stated. The results are shown in Table 15.Furthermore, the isolation of Streptococcus and Actinomyces DNA is shownin FIGS. 9A and 9B.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

TABLE 1 Primers and probes Primers or Probe Sequence (5′-3′) T_(m)(° C.) Universal TCCTACGGGAGGCAGCAGT [SEQ ID NO:1] 59.4 forward primerUniversal GGACTACCAGGGTATCTAATCCTGTT [SEQ ID NO:2] 58.1 reverse primerUniversal probe [6-FAM]CGTATTACCGCGGCTGCTGGCAC[TAMRA] [SEQ ID NO:3] 69.9B. tryoni GGAAGGTAAGTTGCATTTCAGCA [SEQ ID 110:4] 59.3 forward primer B.tryoni GCGTACTTATCATGGTAAATTAAGTCAATT [SEQ ID NO :5] 58.6 reverse primerB. tryoni [VIC]-TCCCGTTACAAAATCGTGTTTACATCGTATACTCG-[TAMRA] [SEQ IDNO:6] 69.1 probe P. gingivalis TCGGTAAGTCAGCGGTGAAAC [SEQ ID NO: 7] 58.8forward primer P. gingivalis GCAAGCTGCCTTCGCAAT [SEQ ID NO: 8] 58.7reverse primer P. gingivalis [6-FAM]CTCAACGTTCAGCCTGCCGTTGAAA[TAMRA][SEQ ID NO:9] 68.8 probe 6-FAM: 6-carboxyfluorescene; TAMRA:6-carboxy-tetramethyirhodamine VIC: Proprietory dye of AppliedBiosystems

TABLE 2 Representative bacterial species^(a) detected by Real-Time PCRusing the universal primer-probe set and estimated bacterial numbersbased on standard graph for E. coli DNA DNA No. of No. of (pg)/bacteria^(b)/ bacteria/ Bacterial species c_(t) 25 μl 25 μl pg DNA^(c)Gram negative aerobic bacteria Pseudomonas aeruginosa 18.14 1240  250 ×10³ 2.02 × 10² ATCC 19660 Legionella pneumophilia 21.93   161.5  33 ×10³ 2.04 × 10² knoxville-1 ATCC 33153 Gram negative facultativeanaerobic bacteria Escherichia coli JM109 19.86 444  89 × 10³ 2.00 × 10²Serratia marsescens 20.96 224  45 × 10³ ATCC 274 Gram negative anaerobicbacteria Porphyromonas gingivalis 23.5   65  13 × 10³ 2.00 × 10² ATCC33277 Prevotella melaninogenica 20.48 364  73 × 10³ 2.00 × 10² ATCC25845 Fusobacterium nucleatum 21.05 262  53 × 10³ 2.02 × 10² ATCC 25586Gram positive bacteria Staphylococcus aureus ATCC 16.15 3975  801 × 10³2.02 × 10² 12600 Streptococcus mutans LT11 18.78 950 192 × 10³ 2.02 ×10² Peptostreptococcus micros 22.83  96  19 × 10³ 1.98 × 10² ATCC 33270Gram positive asporogenous bacteria Lactobacillus acidophilus 20.73 312 63 × 10³ 2.02 × 10² ATCC 4356 Actinomyces Actinomyces israelii ATCC26.38  13  2.6 × 10³  2.00 × 10² 12102 Mycobacterium tuberculosis 26   11  2.2 × 10³  2.00 × 10² H37RV ^(a)Order of microbes is based onBergy's Manual of Determinative Bacteriology (12). ^(b)Estimated fromthe theoretical value: 0.496 picogram E. coli DNA = 100 E. coli cells.^(c)No. of bacteria/pg DNA remains constant for almost all the bacteriasince all values are interpolated from the standard graph using E. coli:DNA. However, based on the size of the genome, no. of bacteria/pg DNAwould differ. Each DNA sample was diluted accordingly to be within theC_(T) range of the standard graph and the mean quantity was estimated bythe machine from the duplicates.

TABLE 3 Specificity of the universal primer-probe set for individualbacterial strains^(a) tested for Real-Time PCR and estimation ofbacterial numbers based on standard graph for E. coli DNA DNA No. of No.of (pg)/ bacteria^(b)/ bacteria/ Bacterial species c_(t) 25 μl 25 μl pgDNA^(c) Gram negative aerobic bacteria Pseudomonas aeruginosa 18.46 1015205 × 10³ 2.02 × 10² ATCC 15442 Pseudomonas aeruginosa 18.26 1130 228 ×10³ 2.02 × 10² 6294 Pseudomonas aeruginosa 19.67 485  98 × 10³ 2.02 ×10² 6206 Pseudomonas fluorescens 19.19 650 131 × 10³ 2.02 × 10²Pseudomonas putida 22.35 98  20 × 10³ 2.04 × 10² Pseudomonas stutzeri18.87 790 159 × 10³ 2.01 × 10² Pseudomonas alcaligens 19.33 615 124 ×10³ 2.02 × 10² Pseudomonas species 19.52 530 107 × 10³ 2.02 × 10²Legionella pneumophila 21.34 223  45 × 10³ 2.02 × 10² serogroup 4 ATCC33156 Legionella pneumophila 20.08 477  96 × 10³ 2.01 × 10² serogroup 5ATCC 33216 Legionella pneumophila 21.19 228  46 × 10³ 2.02 × 10²serogroup 6 ATCC 33215 Legionella pneumophila 25.18 25  5.1 × 10³  2.04× 10² philadelphia-1 ATCC 33152 Legionella anisa 24.02 49  9.9 × 10³ 2.02 × 10² Legionella bozemanii 21.46 209  42 × 10³ 2.01 × 10² serogroup2 Legionella londiniensis 20.5  361  73 × 10³ 2.02 × 10² Legionellamacearchernii 22.97 90  18 × 10³ 2.00 × 10² Legionella waltersii 21.96158  32 × 10³ 2.02 × 10² Gram negative facultative anaerobic bacteriaEscherichia coli NM5222 28.22 2.9 0.59 × 10³  2.03 × 10² Escherichiacoli XL 1 blue 26.95 6.3  1.3 × 10³  2.06 × 10² Gram negative anaerobicbacteria Porphyromonas endodontalis 22.05 148  30 × 10³ 2.02 × 10² ATCC35406 Fusobacterium necrophorum 23.15 81  16 × 10³ 1.98 × 10² ATCC 252Gram positive bacteria Staphylococcus aureus ATCC 29.57 1.31 0.27 × 10³ 2.06 × 10² 9144 Staphylococcus aureus ATCC 27.41 4.77 0.96 × 10³  2.01 ×10² 12598 Staphylococcus aureus ATCC 26.32 13  2.7 × 10³  2.07 × 10² BM10458 Staphylococcus aureus ATCC 27.20 5.35  1.1 × 10³  2.05 × 10² BM10143 Staphylococcus epidermidis 17.88 1405 2.83 × 10³  2.01 × 10² ATCC35983 Staphylococcus epidermidis 22.27 102  21 × 10³ 2.05 × 10² ATCC14990 Staphylococcus hemolyticus 21.14 201  41 × 10³ 2.04 × 10² ATCC29970 Staphylococcus hemolyticus 22.28 112.5  23 × 10³ 2.04 × 10²Staphylococcus schleferi 22.29 102  21 × 10³ 2.05 × 10² ATCC 43808Streptococcus sanguis H1 17.05 2495 503 × 10³ 2.01 × 10² Streptococcussaltvarlus 20.27 410  83 × 10³ 2.02 × 10² Streptococcus gordonii 20.03466  94 × 10³ 2.02 × 10² Peptostreptococcus 22.36 125  25 × 10³ 2.00 ×10² anaerobius ATCC 27337 Gram positive asporogenous bacteriaLactobacillus rhamnosus 24.53 37  7.5 × 10³  2.02 × 10² ATCC 7469Actinomyces Actinomyces neslundii ATCC 24.32 42  8.4 × 10³  2.00 × 10²12104 ^(a)Order of microbes is based on Bergy's Manual of DeterminativeBacteriology (12). ^(b)Estimated from the theoretical value: 0.496picogram E. coli DNA = 100 E. coli cells. Each DNA sample was dilutedaccordingly within the c_(t) range of the standard graph. ^(c)No. ofbacteria/pg DNA remains constant for almost all the bacteria since allvalues are interpolated from the standard graph using E. coli DNA.However, based on the size of the genome, no. of bacteria/pg DNA woulddiffer. Each DNA sample was diluted accordingly to be within the C_(T)range of the standard graph and the mean quantity was estimated by themachine from the duplicates.

TABLE 4 Effect of species specific DNA standards on the relativeestimation of [DNA] using the universal primers-probe set for Real-TimePCR Relative amount of DNA (%)* S. aureus DNA E. coli DNA P. aeruginosaDNA P. endodontalis P. melaninogenica Bacterium A₂₆₀ nm† standardstandard standard DNA standard DNA standard S. aureus 100 106 145 2941231  2600‡ E. coli 100 46 96 139 550 1415 P. aeruginosa 100 48 96 139456  688 P. endodontalis 100 8 17 9 108  193 P. melaninogenica 100 5 1110 68  110 *The species specific standard DNA graphs (c_(t) vs [DNA])were generated from E. coli DNA within the range 238 fg–2.38 ng, from P.aeruginosa DNA within the range 25 fg–2.5 ng, from S. aureus DNA withinthe range 27.5 fg–2.75 ng, from P. melaninogenica DNA within the range1.12 pg–112 ng and from P. endodontalis DNA within the range 240 fg–24ng. The meanof duplicate determinations are shown. Variation betweenduplicates was ≦2.7% except where underlined where the values for the E.coli and P. melaninogenica DNA standard varied by 4.8% and that for theP. aeruginosa DNA standard by 15.9%. †The concentration of DNA wasdetermined spectrophotometrically and normalized to 100% prior todiluting in the range of 100- to 1000-fold for determination byReal-Time PCR. ‡Value halved from that determined by computer software(for explanation, see text).

TABLE 5 Enumeration of bacterial cell numbers by viable cell count andReal-Time PCR. Relative estimation of cell Viable. cell count* numbersby Real-Time PCR† Bacterial Culture [cells (ml culture)⁻¹] [cells (mlculture)⁻¹] E. coli 6.5 × 10⁸  6.7 × 10⁸ P. aeruginosa 3.3 × 10⁹  4.2 ×10⁹ S. aureus 1.3 × 10⁸  2.5 × 10⁹ Mixed culture‡ 1.5 × 10^(9§) 1.3 ×10⁹ *The data are the means of duplicate determinations. Variationbetween duplicates was ≦5.2%. †Based on a standard graph generated by E.coli DNA within the range 238 fg-2.38 ng. The mean of duplicatedeterminations for each of two dilutions of DNA are shown. Variationbetween duplicates did not exceed 3.0% except for one dilution of theunderlined where the variation was 8.8%. ‡The mixed culture consisted ofequal volumes of E. coli, P. aeruginosa and S. aureus cultures.^(§)Estimated from the viable cell numbers measured in each of the threecultures.

TABLE 6 Real-Time PCR estimation of anaerobic bacteria in cariousdentine compared with the total viable anaerobic load* Estimation ofGram-negative bacteria Viable colony by Real-Time PCR† forming units‡Ratio§ Sample [cells (mg dentine)⁻¹] [CFU (mg dentine)⁻¹] [cells/CFU]  13.4 × 10⁸ 9.0 × 10⁶ 38  2 4.5 × 10⁸ 5.5 × 10⁶ 82  3 4.8 × 10⁸ 9.8 × 10⁶49  4 1.3 × 10⁸ 4.8 × 10⁶ 27  5 3.8 × 10⁸ 1.2 × 10⁷ 32  6 5.5 × 10⁸ 1.2× 10⁷ 46  7 1.4 × 10⁸ 6.9 × 10⁶ 21  8 1.1 × 10⁸ 2.0 × 10⁶ 55  9 1.9 ×10⁸ 1.5 × 10⁷ 13 10 3.7 × 10⁸ 2.2 × 10⁷ 17 11 1.4 × 10⁸ 3.1 × 10⁶ 45 123.6 × 10⁸ 5.9 × 10⁶ 61 13 1.5 × 10⁸ 2.2 × 10⁶ 68 14 1.1 × 10⁹ 1.2 × 10⁷92 15 2.6 × 10⁸ 1.4 × 10⁷ 19 16 2.5 × 10⁸ 1.5 × 10⁷ 17 17 2.8 × 10⁸ 8.2× 10⁶ 34 18 6.5 × 10⁸ 1.6 × 10⁷ 41 19 2.5 × 10⁸ 5.6 × 10⁶ 45 20 6.7 ×10⁸ 3.7 × 10⁷ 18 *The method of DNA extraction lyses anaerobicGram-negative and Gram-positive bacteria, but not facultativeGram-positive bacteria. †Based on a standard graph generated by P.melaninogenica DNA within the range 82.9 fg-8.29 ng where 2.36 fg P.melaninogenica DNA represents one cell. The data are the means oftriplicate determinations. The standard deviation of the means varied by<1.0% except for the underlined where the variation was in the range of1.7-4.4%. ‡The data are the means of duplicate determinations. Variationbetween duplicates was <10.0%. §The ratio represents the n-fold increasein anaerobic bacteria detected by Real-Time PCR over the total colonycount which includes facultative Gram-positive bacteria.

TABLE 7 C_(T) value C_(T) value Sample Conditions P. gingivalis DNAInternal positive control Freeze/thaw 24   26.2 Freeze/boil 16   16.6Enzymatic 21.5 20.4 Enzymatic + 5 mM Zn 16.5 17.2 Cl2 10 mM phosphate +5 mM Zn Cl2

TABLE 8 Isolation of P. gingivalis DNA in the presence of InternalPositive Control (IPC-BT)^(a) C_(T) ^(b) value (FAM)^(c) C_(T) ^(b)value (VIC)^(d) DNA isolation P. gingivalis IPC-BT method DNAMultiplex^(e) DNA Multiplex^(e) Freeze-thaw 23.52 22.6 27.6 27.6Freeze-boil 16.3  16.6 16.3 15.9 Enzymatic 20.9  19.8 21.6 21.9Enzymatic + ZnCl₂ 16.05 15.5 16.8 16   ^(a)Input value of the InternalPositive Control (IPC-BT) was at C_(T): 16 ^(b)Threshold cycle: HigherC_(T) values indicates low amount of DNA and lower C_(T) indicates highamount of DNA ^(c)Only reporter dye FAM is read ^(d)Only reporter dyeVIC is read ^(e)Same PCR reaction-well contained the primers and probesets for P. gingivalis as well as IPC-BT

TABLE 9 Estimation of DNA following extraction in ATL buffer from QIAmpDNA Mini Kit (Real-Time PCR quantification) Bacteria Amount of DNA (pg)Fusobacterium nucleatum 507 Porphyromonas endodontalis 251 Porphyromonasgingivalis 921 Prevotella melaninogenica 270 Peptostreptococcus micros83.8 Streptococcus mutans 41.2 Lactobacillus acidophilus 25.0Actinomyces israelii 0.269

TABLE 10 Estimation of DNA following one-step DEPC method (Real-Time PCRquantification) Amount of DNA (pg) Bacteria Absence of DEPC: Presence ofDEPC: Fusobacterium nucleatum 457 295 Porphyromonas endodontalis 255 193Porphyromonas gingivalis 8.59 371 Prevotella melaninogenica 114 124Peptostreptococcus micros 63.9 18.2 Streptococcus mutans 708 550Lactobacillus acidophilus 115 76.7 Actinomyces israelii 1.83 1.53

TABLE 11 Comparison of viable count of P. gingivalis and S. mutans cellswith relative amount of cells estimated by Real-Time PCR and number ofcells calculated based on DNA measurement at A260 as a measure ofrecovery of DNA Relative number of cells^(b) per ml based on Real-Number of cells per ml Time PCR based on A₂₆₀ Viable One-step One-stepcount^(a) per ATL DEPC ATL DEPC Culture ml method method method methodP. gingivalis 1.75 × 10⁸ 4.1 × 10⁹ 3.4 × 10⁹ 4.8 × 10⁹ 5.6 × 10⁹One-step One-step DEPC method DEPC method S. mutans  5.4 × 10⁹ 6.0 × 10⁹9.3 × 10⁹ ^(a) P. gingivalis culture grown on CDC agar plate underanaerobic conditions and S. mutans culture grown on BHI agar plate under5% CO₂. ^(b)Using P. gingivalis DNA as a standard graph (3600 pg to 0.36pg range) considering 100 P. gingivalis cells = 0.250 pg DNA and 100 S.mutans cells = 0.237 pg DNA.

TABLE 12 Estimation of DNA in mixed bacterial culture followingextraction by one-step DEPC method (Real-Time PCR quantification) Amountof DNA (pg) Amount of DNA (pg) reaction using reaction using P.Universal primers-probe gingivalis primers-probe Absence PresenceAbsence Presence Bacteria of DEPC: of DEPC: of DEPC: of DEPC:Fusobacterium 107 392 35.9 188 nucleatum + Porphyromonas gingivalisPrevotella  90 323 44.2 232 melaninogenica + Porphyromonas gingivalisStreptococcus 474 493 59.4 249 mutans + Porphyromonas gingivalis

TABLE 13 Estimation of DNA following extraction by two-step DEPC method(Real-Time PCR quantification) Amount of DNA (pg) following extractionSonicated Continuously Bacteria On ice with pulse sonicatedFusobacterium nucleatum 319 276 123 Porphyromonas endodontalis 198 153122 Porphyromonas gingivalis 327 312 410 Prevotella melaninogenica 58.382.2 67.8 Peptostreptococcus micros 66.7 59.4 64.7 Streptococcus mutans471 437 361 Lactobacillus acidophilus 85.5 80.5 44.4 Actinomycesisraelii 2.47 4.74 3.01

TABLE 14A Relative estimation of P. gingivalis cells and total bacteriain diseased site plaque sample Relative No. of cells estimated per ml ofplaque sample Condition Plaque No. P. gingivalis Total load % P.gingivalis Diseased site 45 1.4 × 10 4.8 × 10¹¹ 29 plaque sample P.gingivalis DNA (3600 pg-0.36 pg) was used for the standard graph forrelative estimation of DNA in the plaque samples. 100 P. gingivaliscells = 0.250 pg DNA.

TABLE 14B Diversity of species in 57 clones analyzed for Sequenced BasedIdentification using 466 bp DN segment amplified using universal primersBacteria No. of species % P. gingivalis 17 29.8 Fusobacteria 18 31.6 B.forsythus 6 10.5 Prevotella 4 7 Treponema 2 3.5 Campylobacter 1 1.8Capnocytophaga 1 1.8 Desufobulbus 1 1.8 Catonella (clostridium) like 11.8 Streptococcus 1 1.8 Clostridium 1 1.8 Porphyromonas like 1 1.8Rothia dentocariosa 1 1.8 Flexistipes like 1 1.8 Uncultured bacterium 11.8

TABLE 15 Sequence based identification of bacteria from dental plaqueflora Culture High score bacterial species % Similarity % Identity 4-2S. mitis SM16SRR1 99.3 99.3 S. costellatus AF104677 94 94 S. anginosusAF306833 94 94 S. intermedius AF104673 94.4 94.4 2-2-1 S. mitis SM16SRR194.5 93.7 S. costellatus AF104677 98.24 97.49 S. anginosus AF30683398.74 97.99 S. intermedius AF104673 99.50 98.74 6-5 S. mitis SM16SRR194.8 94.5 S. costellatus AF104677 98.7 98.4 S. anginosus AF306833 99.298.96 S. intermedius AF104673 100 99.74 10-34 S. mitis SM16SRR1 94.5694.3 S. costellatus AF104677 100 99.74 S. anginosus AF306833 98.45 98.19S. intermedius AF104673 98.7 98.45 1-2-1 Actinomyces species oral clone99.24 98.49 AF385553 A. viscosus AVRRNA16S 98.99 98.24 A. naeslundiiANE234051 98.995 97.99 A. meyeri AMRNAR16S 92.68 91.92 A. georgiaeAG16SRRNA 92.93 92.17 A. odontolyticus AOD234041 91.41 90.68 4-2-1Actinomyces species oral clone 93.42 93.16 A. viscosus AVRRNA16S 93.1692.90 A. naeslundii ANE234051 93.95 93.42 A. meyeri AMRNAR16S 98.4298.16 A. georgiae AG16SRRNA 99.74 99.47 A. odontolyticus AOD234041 97.6397.37

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1. A method for determining total microbial content in a sample, saidmethod comprising amplifying a target sequence selected from 16S rDNAand 16S rRNA using as a forward primer, an oligonucleotide comprisingthe sequence set forth in SEQ ID NO: 1 and using as a reverse primer, anoligonucleotide comprising the sequence set forth in SEQ ID NO: 2, saidamplification being for a time and under conditions sufficient togenerate a level of an amplification product which is proportional tothe level of microorganisms in said sample.
 2. The method of claim 1wherein the amplification product is detected by hybridizing a probe toa nucleotide sequence nested between said forward and reverse primers.3. The method of claim 1 or 2 wherein the target sequence is 16S rDNA.4. The method of claim 1 or 2 wherein the target sequence is 16S rRNA.5. The method of claim 1 or 2 wherein the sample is a biological,medical, agricultural, industrial or environmental sample.
 6. The methodof claim 5 wherein the medical sample is a culture fluid, biopsy fluidor tissue, swab or sample from oral cavity or other sample.
 7. Themethod of claim 5 wherein the biological sample is from an animal orinsect or plant.
 8. The method of claim 7 wherein the medical sample isfrom an oral cavity.
 9. The method of claim 5 wherein the sample is anenvironmental sample.
 10. The method of claim 9 wherein theenvironmental sample is from soil, river, hot mineral springs, plant,antarctic, air or extraterrestrial samples as well as samples fromindustrial sites such as waste sites and areas of oil spills or aromaticor complex molecule contamination and pesticide contamination.
 11. Themethod of claim 5 wherein the sample comprises food, food components,food derivatives and/or food ingredients including food products formedin the dairy industry such as milk.
 12. The method of claim 5 whereinthe sample is liquid, solid, slurry, air, vapour, droplet, aerosol or acombination thereof.
 13. A method according to claim 1 wherein theamplification is by polymerase chain reaction (PCR).
 14. The method ofclaim 1 or 2 wherein the amplification is by Real-Time PCR.
 15. A methodfor identifying a microorganism by its genus in a sample, said methodcomprising subjecting DNA in said sample to Real-Time PCR using aprimers-probe set which comprises primers selected to amplify DNAcomprising 16S rDNA and a probe which hybridizes to a nucleotidesequence nested between said primers wherein said probe is eitherspecific for said microorganism to be identified or which issubsequently identified by a genus-specific probe wherein the primerscomprise a forward primer comprising the sequence set forth in SEQ IDNO: 1 and a reverse primer comprising the sequence set forth by SEQ IDNO:2.
 16. The method of claim 15 wherein the amplified DNA is 16S rDNA.17. The method of claim 15 wherein the genus-specific probe is also aspecies-specific probe.
 18. The method of claim 15 or 17 wherein theprobe is a polynucleotide having the sequence set forth in SEQ ID NO: 3.19. The method of claim 15 wherein the 16S rDNA is amplified.
 20. Themethod of claim 15 wherein the sample is a biological, medical,agricultural, industrial or environmental sample.
 21. The method ofclaim 20 wherein the medical sample is a culture fluid, biopsy fluid ortissue, swab or sample from oral cavity or other sample.
 22. The methodof claim 20 wherein the biological sample is from an animal or insect orplant.
 23. The method of claim 20 wherein the medical sample is from anoral cavity.
 24. The method of claim 20 wherein the sample is anenvironmental sample.
 25. The method of claim 24 wherein theenvironmental sample is from soil, river, hot mineral springs, plant,antarctic, air or extraterrestrial samples as well as samples fromindustrial sites such as waste sites and areas of oil spills or aromaticor complex molecule contamination and pesticide contamination.
 26. Themethod of claim 20 wherein the sample comprises food, food components,food derivatives and/or food ingredients including food products formedin the dairy industry such as milk.
 27. The method of claim 20 whereinthe sample is liquid, solid, slurry, air, vapour, droplet, aerosol or acombination thereof.
 28. The method of claim 2 wherein the probe is apolynucleotide having the sequence set forth in SEQ ID NO: 3.